Methods and compositions for theranostic nanoparticles

ABSTRACT

Disclosed are compositions and methods for identifying a solid tumor cell target. Compositions and methods for treating prostate cancer are also disclosed. Further, cancer therapeutic compositions comprising CT20p are disclosed. Nanoparticles that are conjugated with a targeting ligand that is a substrate for a solid tumor-specific cell protein are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/153,912, filed Apr. 28, 2015, which is incorporatedby reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersK01CA101781 and GM083324 awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD

The subject matter disclosed herein is in the field of nanoparticles,including methods of identifying and monitoring tumor cells by providinga nanoparticle functionalized with one or more ligands and one or moreimaging compounds.

BACKGROUND

The imaging, diagnosis, and successful treatment of prostate cancer(PCa) continue to be a challenging problem and it is estimated that 1out of 6 men will be diagnosed with the disease during their lifetime.Early detection using existing techniques is difficult due to the (1)relatively small size of the prostate gland, (2) low metabolic rate ofPCa and (3) close proximity of the prostate to the bladder, which limitsthe use of traditional PET imaging using small molecule (¹⁸F-FDG)radionucleotides that accumulate in the bladder before excretion.Meanwhile, current treatment options for PCa, such as surgery, systemicchemotherapy and radiation therapy are often ineffective and usuallyresult in severe side effects for the patients. Therefore, developmentof more effective agents against advanced PCa that allow forsimultaneous therapy and monitoring are urgently needed. Particularlyneeded are targeted molecular theranostic (dual therapy and diagnostic)regimes that allow delivery of a new generation of imaging andtherapeutic agents in high concentrations to PCa.

Death due to prostate cancer (PCa) generally results when patientsdevelop metastatic castration-resistant prostate cancer (mCRPC). Whilecurrent treatments for mCRPC improve survival, the disease still remainsincurable, and treatments result in severe side effects, such asimpotence and incontinence. Current methods to detect PCa and monitortreatment out comes are typically invasive, indicating a need for newimaging agents that use sensitive molecular imaging technologies such asPET (positron emission tomography).

Thus, there is a need for compositions and methods for the delivery andmonitoring of therapeutic peptides to areas of disease. These needs andother needs are satisfied by the present invention.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, articles, devices, and methods, as embodied and broadlydescribed herein, the disclosed subject matter relates to compositionsand methods of making and using the compositions. In other aspects, thedisclosed subject matter relates to nanoparticles comprising a polymericnanoparticle conjugated with targeting ligand that is a substrate for asolid tumor-specific cell protein, wherein the nanoparticle furthercomprises an imaging compound and has a therapeutic agent encapsulatedin the hydrophobic interior of the nanoparticle. A cancer therapeuticcomposition comprising the nanoparticle are also disclosed.

In a further aspect, disclosed herein are methods of identifying a solidtumor cell target comprising contacting a cell with an effective amountof a composition comprising the nanoparticles disclosed herein.

In a still further aspect, disclosed herein is a method for treatingprostate cancer, comprising administering to a subject diagnosed withprostate cancer an effective amount of the nanoparticle composition.

Additional advantages of the disclosed subject matter will be set forthin part in the description that follows and the Figures, and in partwill be obvious from the description, or can be learned by practice ofthe aspects described below. The advantages described below will berealized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figure, which is incorporated in and constitutes a partof this specification, illustrates several aspects and together with thedescription serves to explain the principles of the invention.

FIG. 1 is a schematic representation of a method for the development andscreening of a multivalent theranostic nanoparticle library for PSMAtargeting.

FIG. 2 depicts the structure of a HBPE polymer.

FIG. 3 depicts the structure of a DFO-Zr-HBPE polymer.

FIG. 4 is a graph showing the cell-associated fluorescence of variouscancer cell lines after treatment with HBPE(DiI)-folate nanoparticles(folate NP, left axis). The presence of PSMA in these cell lines wascorroborated using the anti-PSMA antibody J590 (right axis).

FIG. 5 shows the targeting of the PSMA receptor in LNCaP prostate cancercells using folate and glutamate-derivatized HBPE nanoparticles.

FIG. 6 depicts the accumulation of HBPE(DiR)-folate nanoparticles inPSMA positive PC3 tumor in mice. Increased uptake was observed for alltime points in the PSMA-positive PC3-PSMA tumor (representative animal).(Light grey indicates high uptake, while dark grey indicates lowuptake).

FIG. 7 depicts the synthetic route for the DFO-HBPE nanoparticle.

FIG. 8 shows the HBPE-DFO:Zr Nanoparticle size distribution determinedby DLS. Insent: corresponding STEM image of the nanoparticles. Scalebar: 100 nm.

FIG. 9A is a graph showing the pH-dependent abiraterone drug release ofHBPE nanoparticles. FIG. 9B is a graph showing the cytotoxicity profileof HBPE nanoparticles (ABE=abiraterone).

FIG. 10 depicts HBPE(DiI) folate and HBPE (DiI) glutamate nanoparticlesthat encapsulate abiraterone induce cell death in LNCaP cells, thatexpress PSMA.

FIG. 11 depicts the general synthetic scheme toward Scaffold 1 analogs.

FIG. 12 depicts the general synthetic scheme toward Scaffold 2-3analogs.

FIG. 13 depicts the general synthetic scheme toward Scaffold 4 analogs.

FIG. 14A depicts a mass spectrometry confirmation of the ability of DFOand DFO:Fe to chelate Zr. FIG. 14B shows the generation of the⁸⁹Zr-DFO-HBPE nanoparticles from Fe-DFO-HBPE.

FIG. 15 is a schematic representation of a proposed mechanism by whichCT20p, in HBPE-NPs, is released from endosomes/lysosomes under acidicconditions, forms a pore, and translocates to the cytosol via chaperoneto bind to mitochondria.

FIG. 16 illustrates the synthetic route for Gd-DTPA andFe(III)-DFO-HBPE-NPs.

FIG. 17A depicts a solvent diffusion method used to fabricate thefolate-HBPE-DFO(CT20p)-NPs. The polymer and CT20p were dissolved in awater-miscible beaker containing water under constant stirring. FIG. 17Bis a representative STEM image of NPs. FIG. 17C is a graph showing theCT20p release profile at acidic pH.

FIGS. 18A-18J illustrate a timeline of CT20p activities in cancer cells.FIG. 18A shows that rhodamine-labeled CT20p (red) co-localizes withmitochondria (mitotracker green). FIG. 18B shows that mitochondrialmembranes hyperpolarize and fuse (JC-1 probe). FIG. 18C shows thatmitochondria (red) fail to redistribute to cell extensions, causingreduced F-actin (green) polymerization (nucleus, DAPI, blue). FIG. 18Dshows that the initial viability of cells was determined by measuringmembrane permeability (Sytox) and membrane asymmetry (violet ratiometricdye). Gates are N, necrotic; V, viable; A, apoptotic. Percentages are V(black) and N+A (red). FIG. 18E is a graph showing that by 6 hours,cells detach from the substrate (fibronectin). Such cell detachment wasmeasured using a crystal violet adhesion assay. FIG. 18F shows thatprior to detecting cell detachment, membrane levels of β1 integrindecreased as detected with an anti-β1 antibody. FIGS. 18G-18I show thatpost-cell detachment events include caspase activation (FIG. 18G: showsdetection of caspase3/7 activity by colorimetric assay), autophagy (FIG.18H: shows the formation of autophagosomes detected by GFP-LC3), andincreased ROS production (FIG. 18I: shows mitochondrial superoxidedetected using Mitosox). FIG. 18J shows that apoptosis/anoikis wasdetected between 24-48 hours as described in FIG. 5D. *p<0.5

FIG. 19A-19C show that normal cells were affected by CT20p. FIG. 19Ashows that rhodamine-labeled CT20p (red) did not co-localize withmitochondria (green) or cause autophagy (no autophagosomes formed). FIG.19B shows results after 24 hours, LC3-GFP. FIG. 19C shows that minimalcell death was detected.

FIG. 20 shows the results of a FACS analysis used to assess the degreeof targeting and PSMA-mediated cell internalization ofFolate.HBPE(DiI)-NPs. Also shown are the corresponding fluorescenceimages.

FIGS. 21A-21J show dose- (FIGS. 21A, 21C) and time- (FIGS. 21D, 21F)dependent cytotoxicity assay of PCa cells treated withFolate.HBPE(Dil)-NPs. PCa Cells: LNCap (FIGS. 21A, 21D), PSMA(+) PC3(FIGS. 21B, 21E) and PC3 (FIGS. 21C, 21F). FIG. 21G shows thefluorescence microscopy image of PSMA(+) PCa cells treated with FolateHBPE(CT20p) NPs and FIG. 21H shows the corresponding Dil fluorescence.FIG. 21I shows the results of the sytox analysis using macrophagesincubated with CT20p (left), doxorubicin (middle), and Folate-s-s-Doxo(right). V, viable; N, necrotic, A, apoptotic.

FIG. 22A depicts an image of mice that were injected subcutaneously (SC)with PSMA(+) (right flank) or PSMA(−) (left flank) PCa tumor cells. Upontumor detection (˜2 weeks), the mice were injected intravenously (IV)with PEG-(FOL)-HBPE-NPs (2 mg/kg/dose) containing a near IR dye (FIG.22A) or CT20p (FIG. 22B). Mice were imaged after 24 hours (FIG. 22A) orsacrificed after 10 days (FIG. 22B). FIG. 22C shows an image of thetissue harvested from FIG. 22B for histological examination. Fragmentedand necrotic tissue in the PSMA+ tumor is indicated by arrow and bordersmarked by a line. FIG. 22D is a graph that summarizes a two weekexperiment in which mice (n=5) bearing PSMA+ tumors (SC) were IVinjected once per week with FOL-HBPE-NPs (2 mg/kg/dose) that were emptyor had CT20p and were compared to COOH-NPs (untargeted) with CT20p orFOL-targeted doxorubicin (DOX). *p<0.05. The mice were euthanized beforethe tumors ulcerated.

FIG. 23 shows the γ- (top) and α- (bottom) polyglutamated acid folatepeptides used herein.

DETAILED DESCRIPTION

The disclosed subject matter can be understood more readily by referenceto the following detailed description, the Figures, and the examplesincluded herein.

Before the present compositions and methods are disclosed and described,it is to be understood that they are not limited to specific syntheticmethods unless otherwise specified, or to particular reagents unlessotherwise specified, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated,it is in no way intended that any method set forth herein be construedas requiring that its steps be performed in a specific order.Accordingly, where a method claim does not actually recite an order tobe followed by its steps or it is not otherwise specifically stated inthe claims or descriptions that the steps are to be limited to aspecific order, it is in no way intended that an order be inferred, inany respect. This holds for any possible non-express basis forinterpretation, including matters of logic with respect to arrangementof steps or operational flow, plain meaning derived from grammaticalorganization or punctuation, and the number or type of aspects describedin the specification.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

It is understood that the disclosed methods and systems are not limitedto the particular methodology, protocols, and systems described as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

Definitions

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, the amino acid abbreviations are conventional one lettercodes for the amino acids and are expressed as follows: A, alanine; B,asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate,glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine;K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

“Peptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. For example,a peptide can be a fragment of a full-length protein, such as, forexample, the CT20 peptide. A peptide is comprised of consecutive aminoacids. The term “peptide” encompasses naturally occurring or syntheticmolecules.

In general, the biological activity or biological action of a peptiderefers to any function exhibited or performed by the peptide that isascribed to the naturally occurring form of the peptide as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). For example, abiological activity of the CT20 peptide is the cytotoxic activity of theCT20 peptide.

The term “enzyme” as used herein refers to any peptide that catalyzes achemical reaction of other substances without itself being destroyed oraltered upon completion of the reaction. Typically, a peptide havingenzymatic activity catalyzes the formation of one or more products fromone or more substrates. Such peptides can have any type of enzymaticactivity including, without limitation, the enzymatic activity orenzymatic activities associated with enzymes such as those disclosedherein.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the terms “transformation” and “transfection” mean theintroduction of a nucleic acid, e.g., an expression vector, into arecipient cell including introduction of a nucleic acid to thechromosomal DNA of said cell. The art is familiar with variouscompositions, methods, techniques, etc. used to effect the introductionof a nucleic acid into a recipient cell. The art is familiar with suchcompositions, methods, techniques, etc. for both eukaryotic andprokaryotic cells. The art is familiar with such compositions, methods,techniques, etc. for the optimization of the introduction and expressionof a nucleic acid into and within a recipient cell.

As used herein, “a CT20 peptide” or “CT20” may refer to one peptide ormay refer one or more peptides (i.e., a C-terminal Bx peptide), such asmolar concentrations of the peptide, as would be found in a composition.In an aspect, a CT20 peptide can comprise SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In an aspect, a CT20peptide can comprise a combination of two or more of SEQ ID NOs:1-6.Those skilled in the art understand where an individual peptide isintended and where a molar, or smaller or larger amount, of many of thesame peptide are intended.

As used herein, “noncancerous cells” and “noncancerous tissue” can referto cells or tissue, respectively, that are normal or cells or tissuethat do not exhibit any metabolic or physiological characteristicsassociated with cancer. For example, noncancerous cells and noncanceroustissues are healthy and normal cells and tissues, respectively.

As used herein, the term “subject” refers to the target ofadministration, e.g., an animal. Thus, the subject of the hereindisclosed methods can be a vertebrate, such as a mammal, a fish, a bird,a reptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a patient. A patient refers to a subjectafflicted with a disease or disorder, such as, for example, cancerand/or aberrant cell growth. The term “patient” includes human andveterinary subjects. In an aspect, the subject has been diagnosed with aneed for treatment for cancer and/or aberrant cell growth.

Therapeutic agents can include antimicrobial agents, such as antibioticsor antimycotic compounds, including but not limited to, active agentssuch as antifungal agents, antibacterial agents, anti-viral agents andantiparasitic agents, and metals. An antimicrobial agent can comprise asubstance, compound or molecule, which kills or inhibits the growth ofmicroorganisms such as bacteria, fungi, or protozoans. Antimicrobialagents may either kill microbes (microbiocidal) or prevent the growth ofmicrobes (microbiostatic). Disinfectants are antimicrobial substancesused on non-living objects or outside the body. Antimicrobial agentsinclude those obtained from natural sources, such as Beta-lactamantibiotics (such as penicillins, cephalosporins), and protein synthesisinhibitors (such as aminoglycosides, macrolides, tetracyclines,chloramphenicol, polypeptides), and those from synthetic sources such assulphonamides, cotrimoxazole, quinolones, anti-fungals, anti-cancerdrugs, anti-malarials, anti-tuberculosis drugs, anti-leprotics, andanti-protozoals.

Examples of antimicrobial agents that can be used herein include, butare not limited to, isoniazid, ethambutol, pyrazinamide, streptomycin,clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin,rifampin, azithromycin, clarithromycin, dapsone, tetracycline,erythromycin, ciprofloxacin, doxycycline, ainpicillin, amphotericin B,ketoconazole, fluconazole, pyrimethaniine, sulfadiazine, clindamycin,lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir,trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir,iatroconazole, miconazole, Zn-pyrithione, heavy metals including, butnot limited to, gold, platinum, silver, zinc and copper, and theircombined forms including, salts, such as chloride, bromide, iodide andperiodate, and complexes with carriers, and other forms. As used herein,the term metal includes all metal salts or metal compounds, including,but not limited to, metal chlorides, metal phosphates, metal sulfates,metal iodides or metal bromides. The active form of some metal salts isthe ionic form. Other antimicrobial agents include, but are not limitedto, polyene antifungals, Amphotericin B, Candicidin, Filipin, Hamycin,Natamycin, Nystatin, Rimocidin, Imidazoles, Bifonazole, Butoconazole,Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole,Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole,Tioconazole, Triazoles, Albaconazole, Fluconazole, Isavuconazole,Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole,Thiazoles, Abafungin, Allylamines, Amorolfin, Butenafine, Naftifine,Terbinafine, Echinocandins, Anidulafungin, Caspofungin, Micafungin.

The terms “treating”, “treatment”, “therapy”, and “therapeutictreatment” as used herein refer to curative therapy, prophylactictherapy, or preventative therapy. As used herein, the terms refers tothe medical management of a subject or a patient with the intent tocure, ameliorate, stabilize, or prevent a disease, pathologicalcondition, or disorder, such as, for example, cancer or a tumor. Thisterm includes active treatment, that is, treatment directed specificallytoward the improvement of a disease, pathological condition, ordisorder, and also includes causal treatment, that is, treatmentdirected toward removal of the cause of the associated disease,pathological condition, or disorder. In addition, this term includespalliative treatment, that is, treatment designed for the relief ofsymptoms rather than the curing of the disease, pathological condition,or disorder; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development of theassociated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder. In various aspects, the term coversany treatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In an aspect, the disease, pathological condition, or disorder iscancer, such as, for example, breast cancer, lung cancer, colorectal,liver cancer, or pancreatic cancer. In an aspect, cancer can be anycancer known to the art.

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed. For example, in an aspect, preventing can refer to thepreventing of replication of cancer cells or the preventing ofmetastasis of cancer cells.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician or aresearcher, and found to have a condition that can be diagnosed ortreated by compositions or methods disclosed herein. For example,“diagnosed with cancer” means having been subjected to a physicalexamination by a person of skill, for example, a physician or aresearcher, and found to have a condition that can be diagnosed ortreated by a compound or composition that alleviates or amelioratescancer and/or aberrant cell growth.

As used herein, the terms “administering” and “administration” refer toany method of providing a peptide (such as a CT20 peptide), or acomposition (such as a composition comprising a CT20 peptide), orpharmaceutical preparation (such as a preparation comprising a CT20peptide or a composition comprising a CT20 peptide) to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, intracardiac administration, oral administration,transdermal administration, administration by inhalation, nasaladministration, topical administration, intravaginal administration,ophthalmic administration, intraaural administration, intracerebraladministration, rectal administration, sublingual administration, buccaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcomposition or peptide or pharmaceutical preparation and a cell, targetreceptor, or other biological entity together in such a manner that thecompound can affect the activity of the target (e.g., receptor,transcription factor, cell, etc.), either directly; i.e., by interactingwith the target itself, or indirectly; i.e., by interacting with anothermolecule, co-factor, factor, or protein on which the activity of thetarget is dependent.

As used herein, the term “determining” can refer to measuring orascertaining a quantity or an amount or a change in expression and/oractivity level, e.g., of a nucleotide or transcript or polypeptide(e.g., CCT or a CCT subunit). For example, determining the amount of adisclosed transcript or polypeptide in a sample as used herein can referto the steps that the skilled person would take to measure or ascertainsome quantifiable value of the transcript or polypeptide in the sample.The art is familiar with the ways to measure an amount of the disclosednucleotides, transcripts, polypeptides, etc.

In an aspect, “determining” as used herein can refer to measuring orascertaining the level of cell death or cell survival, for example,following administration of a CT20 peptide or a composition comprisingan effective amount of a CT20 peptide. Methods of measuring orascertaining cell survival and cell death are known to the art andinclude, but are not limited to, histochemical staining (e.g., TUNEL),cell proliferation assay, cell death assays, morphological examination,etc. In an aspect, the size of a tumor can be measured non-invasivelythrough, for example, ultrasound or imaging.

As used herein, the term “level” refers to the amount of a targetmolecule in a sample, e.g., a sample from a subject. The amount of themolecule can be determined by any method known in the art and willdepend in part on the nature of the molecule (i.e., gene, mRNA, cDNA,protein, enzyme, etc.). The art is familiar with quantification methodsfor nucleotides (e.g., genes, cDNA, mRNA, etc.) as well as proteins,polypeptides, enzymes, etc. It is understood that the amount or level ofa molecule in a sample need not be determined in absolute terms, but canbe determined in relative terms (e.g., when compare to a control or asham or an untreated sample).

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, in an aspect,an effective amount of a CT20 peptide is an amount that kills and/orinhibits the growth of cells without causing extraneous damage tosurrounding non-cancerous cells. For example, a “therapeuticallyeffective amount” refers to an amount that is sufficient to achieve thedesired therapeutic result or to have an effect on undesired symptoms,but is generally insufficient to cause adverse side effects. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration; the route of administration; therate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed and like factors well known in the medical arts.

By “modulate” is meant to alter, by increase or decrease. As usedherein, a “modulator” can mean a composition that can either increase ordecrease the expression level or activity level of a gene or geneproduct such as a peptide. Modulation in expression or activity does nothave to be complete. For example, expression or activity can bemodulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or any percentage in between as compared to a control cellwherein the expression or activity of a gene or gene product has notbeen modulated by a composition.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a CT20 peptide or a disclosed compositioncomprising a CT20 peptide) that is required for 50% inhibition ordiminution of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. IC₅₀ also refersto the concentration or dose of a substance that is required for 50%inhibition or diminution in vivo, as further defined elsewhere herein.Alternatively, IC₅₀ also refers to the half maximal (50%) inhibitoryconcentration (IC) or inhibitory dose of a substance. The response canbe measured in an in vitro or in vivo system as is convenient andappropriate for the biological response of interest. For example, theresponse can be measured in vitro using cultured cancer cells or in anex vivo organ culture system with isolated cancer cells (e.g., breastcancer cells, pancreatic cancer cells, liver cancer cells, lung cancercells, colorectal cancer cells, etc.). Alternatively, the response canbe measured in vivo using an appropriate research model such as rodent,including mice and rats. The mouse or rat can be an inbred strain withphenotypic characteristics of interest such as, for example, cancerand/or aberrant cell growth. As appropriate, the response can bemeasured in a transgenic or knockout mouse or rat wherein a gene orgenes has been introduced or knocked-out, as appropriate, to replicate adisease process.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner. As used herein, the term “pharmaceuticallyacceptable carrier” refers to sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. Thesecompositions can also contain adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents. Prevention of theaction of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents such as paraben, chlorobutanol,phenol, sorbic acid and the like. It can also be desirable to includeisotonic agents such as sugars, sodium chloride and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents, such as aluminum monostearate and gelatin,which delay absorption. Injectable depot forms are made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use. Suitable inert carriers can include sugars suchas lactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers.

As used herein, the term “cancer” refers to a proliferative disorder ordisease caused or characterized by the proliferation of cells which havelost susceptibility to normal growth control. The term “cancer” includestumors and any other proliferative disorders. Cancers of the same tissuetype originate in the same tissue, and can be divided into differentsubtypes based on their biological characteristics. Cancer includes, butis not limited to, melanoma, leukemia, astrocytoma, glioblastoma,lymphoma, glioma, Hodgkin's lymphoma, and chronic lymphocyte leukemia.Cancer also includes, but is not limited to, cancer of the brain, bone,pancreas, lung, liver, breast, thyroid, ovary, uterus, testis,pituitary, kidney, stomach, esophagus, anus, and rectum.

As used herein, the term “sensitizing” refers to an increasedsensitivity of a cell or a subject to a treatment, such as a therapeutictreatment. The term “sensitizing” also refers to a reduction or decreasein the resistance of a cancer cell or a subject with cancer inresponding to a therapeutic treatment. An increased sensitivity or areduced sensitivity to a therapeutic treatment is measured according toa known method in the art for the particular treatment and methodsincluding, but not limited to, cell proliferation assays and cell deathassays. The sensitivity or resistance may also be measured in a subjectby measuring the tumor size reduction over a period of time, such as,for example, every 1 to 3 to 6 month for a human subject and every 2 to4 to 6 weeks for non-human subject (e.g., mouse or rat). The sensitivityof a cell or a subject to treatment can be measured or determined bycomparing the sensitivity of a cell or a subject followingadministration of a CT20 peptide or a composition comprising aneffective amount of a CT20 peptide to the sensitivity of a cell orsubject that has not been administered a CT20 peptide or a compositioncomprising an effective amount of a CT20 peptide.

As used herein, the term “anti-cancer” or “anti-neoplastic” drug refersto one or more drugs that can be used in conjunction with a CT20 peptideor a composition comprising an effective amount of a CT20 peptide totreat cancer and/or aberrant cell growth. Examples of anti-cancer drugsor anti-neoplastic drugs include, but are not limited to, the following:Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin;Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine;Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; InterferonAlfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; ToremifeneCitrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride.

Other anti-neoplastic compounds include: 20-epi-1,25 dihydroxyvitaminD3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; fmasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance genieinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; propylbis-acridone; prostaglandin J2; proteasome inhibitors; protein A-basedimmune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfmosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene dichloride; topotecan; topsentin; toremifene; totipotent stemcell factor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;zinostatin stimalamer.

As used herein, radiosensitizers make a cancer cell more likely to bedamaged. Radiosensitizers enhance the sensitivity of cancer cells and/ora tumor to ionizing radiation, thereby increasing the efficacy ofradiotherapy. Examples of radiosensitizers include gemcitabine,5-fluorouracil, pentoxifylline, and vinorelbine.

The majority of chemotherapeutic drugs can be divided in to: alkylatingagents (e.g., cisplatin, carboplatin, oxaliplatin, mechloethamine,cyclophosphamide, chlorambucil), anti-metabolites (e.g., azathioprine,mercaptopurine), anthracyclines, plant alkaloids and terpenoids (e.g.,vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,and podophyllotoxin) and taxanes (e.g., paclitaxel and docetaxel),topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate, and teniposide), monoclonal antibodies(e.g., trastuzumab, cetuximab, rituximab, bevacizumab), other antitumouragents (e.g., dactinomycin), and hormonal therapy (e.g., steroids suchas dexamethasone, finasteride, aromatase inhibitors, andgonadotropin-releasing hormone agonists).

Disclosed are the components to be used to prepare a compositiondisclosed herein as well as the compositions themselves to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions disclosed herein. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods disclosedherein.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The disclosed subject matter can be practiced in theabsence of any element or elements, limitation or limitations that arenot specifically disclosed herein. Thus, for example, in each instanceherein any of the terms “comprising”, “consisting essentially of”, and“consisting of” can be replaced with either of the other two terms,while retaining their ordinary meanings. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by embodiments, optional features,modification and variation of the concepts herein disclosed can beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

Nanoparticles

A nanoparticle-based therapeutics is ideal as a single agent delivers adrug and imaging agent to the prostate tumor via recognition of surfacereceptor markers highly expressed on the tumor cells. The prostatespecific membrane antigen (PSMA) is a type II transmembrane glycoproteinwith glutamate carboxylase and folate hydrolase activity, highlyexpressed in PCa. PSMA expression usually increases with PCa progressionand metastasis, providing an excellent target for PCa detection andtreatment, especially for the more aggressive forms of the disease. Inaddition, high levels of PSMA have been found on the endothelial cellsof the tumor-associated neovasculature of other solid tumors, includingbreast, lung, colon and pancreas, but not on the normal vasculature.

PSMA exhibits an enzymatic function as a folate hydrolase, hydrolyzingextracellular polyglutamated folate to mono-glutamic folic acid that canthen be utilized by cells. It has been proposed that upregulation ofPSMA can provide PCa cells with a growth advantage in a low folate tumormicro-environment and implicate PSMA in the metabolism of polyglutamatedfolates and the subsequent uptake of folates. Folic acid, a highaffinity ligand for the folate receptor (FR), retains its receptorbinding and endocytosis properties when covalently linked to a widevariety of molecules and nanoparticles. Liposome conjugated folateligands have been used for the delivery of drugs to FR-bearing tumors.However, the use of folate and polyglutamated folate ligands to deliverchemotherapeutics or nanoparticles to PSMA-bearing PCa tissues and theneovasculature of many other tumors had not been studied in detail. Theexperiments disclosed herein took advantage of PSMA's binding affinitytowards polyglutamated folate molecules and developed a library ofnanoparticles conjugated with polyglutamated folate derivatives totarget PSMA. The experiments developed multifunctional, multimodal andmultivalent nanoparticle systems that are used to simultaneously deliverimaging agents and potent anti-androgenic drugs specifically to PCa viaPSMA targeting. The specific targeting of these nanoagents to PCareduced the drugs' systemic exposure, and its associated imagingfunction facilitated in vivo imaging to assess drug delivery to thetumor.

PSMA has already been used to target imaging and therapeutic agents toPCa. Anti-PSMA monoclonal antibody (mAb) has been developed to image anddeliver chemotherapeutics directly to PCa with suboptimal results andlow sensitivity to detect viable tumors. However, high manufacturingcosts limit their widespread application for the targeting and treatmentof tumors. Aptamers have also been investigated as an alternative toantibodies. PSMA-binding aptamers have been identified and conjugated topolymeric nanoparticles encapsulating the anticancer drug docetaxel forthe targeted treatment of LNCaP xenografts in nude mice. However, thesestudies have not been reproducible due to stability issues with theaptamers in serum. Even though, antibodies and aptamers have beenconjugated to polymeric nanoparticles to target PSMA in the past, andsome of these nanoparticle formulations are currently in Phase Iclinical trials, these nanoparticles do not possess imagingcapabilities. Furthermore, the effect of ligand multivalency on thesenanoparticle formulations and the effect on targeting ability have notbeen studied. The ligand's density on the nanoparticle's surface plays akey role in target recognition, specificity and sensitivity in in vitrodiagnostic assays and also plays a role in vivo. Disclosed herein arecompositions and methods that provide insight on the role ofmultivalency in the in vivo delivery of therapeutics and imaging agents.In addition, the compositions and methods used herein are significantlydifferent from the ones previously investigated since small moleculesare utilized, not PSMA targeting aptamers or anti-PSMA monoclonalantibodies which are costly and difficult to make. Finally, as PSMA isalso expressed in the neovasculature of other solid tumors, thecompositions and methods disclosed herein are used on other types ofcancers besides PCa by targeting PSMA expression on the tumorneovasculature and not the tumor itself.

The current disclosure comprises design and fabrication of polymericnanoparticles capable of displaying targeting ligands (polyglutamatedfolates) at high and low density. A rationally-designed compound libraryof ligands containing folic and glutamic acid functionalities wassynthesized and conjugated to the nanoparticles at high and low densitywith the goal of identifying a particular ligand-nanoparticle conjugatethat specifically binds to PSMA in PCa. These nanoparticles conjugateswere used to study the effect of multivalency on PSMA targeting usingpolyglutamated folate ligands. Next, members of the nanoparticle librarywith the most specific binding to PSMA in cell culture were used inanimal studies for the delivery of potent antiandrogenic drugs and a PETtracer (⁸⁹Zr) to PCa via PSMA targeting (FIG. 1).

Thus, disclosed herein are nanoparticles. In an aspect, thenanoparticles are hyberbranched polyester polymeric nanoparticles(HBPE-NPs or just HBPE). In an aspect, the nanoparticles are polymericnanoparticles. In an aspect, the nanoparticles can comprise afunctionalizing group that can be used to attach targeting ligands,therapeutics, or imaging agents. Examples of suitable functionalizinggroups that can be present on the disclosed nanoparticles are azides,amines, alcoholds, esters, and the like. In a specific aspect, disclosedare HBPE nanoparticles with these functionalizing groups, in particularazides. In an aspect, the nanoparticles can comprise a targeting moiety.In an aspect, the nanoparticles are conjugated with one or moretargeting ligands. In an aspect, the targeting ligand is a folatecompound. In an aspect, the targeting ligand is a glutamate compound. Inan aspect, the targeting ligand is a polyglutamated folate compound. Inan aspect, the targeting ligand is glutamate azido urea. In an aspect,the targeting ligand is folate azido urea. In an aspect, the targetingligand is glutamate azido urea. In an aspect, the targeting ligand is abifunctional glutamate-folate hybridized compound. In an aspect, thetargeting ligand is at high density. In an aspect, the targeting ligandis at low density. In an aspect, the targeting ligand is at highvalency. In an aspect, the targeting ligand is at low valency. In anaspect, the targeting ligand is a substrate for a solid tumor-specificcell protein. In an aspect, the solid tumor-specific cell protein isprostate specific membrane antigen (PSMA).

In an aspect, the nanoparticles comprise an imaging compound. In aspect,the imaging compound is a PET detectable compound. In an aspect, the PETdetectable compound is ⁸⁹Zr. In an aspect, the PET detectable compoundis CU or other PET detectable compounds.

In another aspect, the nanoparticles comprise one or more therapeuticagents that are encapsulated in the hydrophobic interior of thenanoparticle. In an aspect, the one or more therapeutic agents areCT20p. In another aspect, the one or more therapeutic agents are mutantCT20 peptides. A CT20 peptide is a C-terminal Bax peptide. Bax is a 21kD protein of 192 amino acids, comprised of nine alpha helices (Suzukiet al., 2000). Under non-apoptotic conditions, Bax predominantly residesin the cytosol, with a small percentage of the protein localized to themitochondria (Boohaker et al., 2011; Kaufmann et al., 2003; Putcha etal., 1999). Bax peptides, Bax proteins, and Bax genes are known to thoseskilled in the art. In an aspect, the one or more therapeutic agents aremitotoxic peptides. In an aspect, the one or more therapeutic agents areanti-metastatic agents. In an aspect, the one or more therapeutic agentsare anti-androgenic agents. In an aspect, the one or more therapeuticagents are anti-neoplastic agents.

In an aspect, the nanoparticles comprise a chelating ligand such asdesferrioxamine (DFO). In an aspect, the nanoparticles arepolyglutamated folate-HBPE-DFO[CT20p]-nanoparticles. In an aspect, thenanoparticle comprises PEG.

Cancer Therapeutic Compositions

Compositions for Dual Targeting and/or Imaging

Disclosed herein are cancer therapeutic compositions. In an aspect, thecancer therapeutic compositions comprise at least one nanoparticle. Inan aspect, the nanoparticles are hyberbranched polyester polymericnanoparticles (HBPE-NPs). In an aspect, the nanoparticles are polymericnanoparticles. In an aspect, the nanoparticles can comprise a targetingmoiety. In an aspect, the nanoparticles are conjugated with a targetingligand. In an aspect, the targeting ligand is a folate compound. In anaspect, the targeting ligand is a glutamate compound. In a specificaspect, the targeting ligand can be an agent that binds to the folatereceptor or the glutamate receptor. In a specific aspect, the targetingligand can be an antibody specific for these receptors, which can beconjugated to the nanoparticle with NHS/EDS or click chemistry (azidefunctional group bonding to a dipolarophile like an alkene or alkyne).In an aspect, the targeting ligand is a polyglutamated folate compound.In an aspect, the targeting ligand is glutamate azido urea. In anaspect, the targeting ligand is folate azido urea. In an aspect, thetargeting ligand is glutamate azido urea. In an aspect, the targetingligand is a bifunctional glutamate-folate hybridized compound. In anaspect, the targeting ligand is at high density. In an aspect, thetargeting ligand is at low density. In an aspect, the targeting ligandis at high valency. In an aspect, the targeting ligand is at lowvalency. In an aspect, the targeting ligand is a substrate for a solidtumor-specific cell protein. In an aspect, the solid tumor-specific cellprotein is prostate specific membrane antigen (PSMA).

In an aspect, the nanoparticles comprise one or more imaging compounds.In aspect, the imaging compound is a PET detectable compound. In anaspect, the PET detectable compound is ⁸⁹Zr. In an aspect, the PETdetectable compound is CU or other PET detectable compounds. In anaspect, the nanoparticles comprise a chelating ligand such asdesferrioxamine (DFO). In an aspect, the nanoparticles arepolyglutamated folate-HBPE-DFO[CT20p]-nanoparticles. In an aspect, thenanoparticle comprises PEG. Further examples of chelating ligands thatcan be used include, but are not limited to,2,2′,2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (DOTA)-based chelators, diethylene triamine pentaacetic acid(DTPA)-based chelators, ethylene diamine tetraacetic acid (EDTA), and aderivative or a combination thereof.

In another aspect, the nanoparticles comprise one or more therapeuticagents that are encapsulated in the hydrophobic interior of thenanoparticle. In an aspect, the one or more therapeutic agents areCT20p. In another aspect, the one or more therapeutic agent are a mutantCT20 peptide. In an aspect, the one or more therapeutic agents are amitotoxic peptide. In an aspect, the one or more therapeutic agents areanti-metastatic agents. In an aspect, the one or more therapeutic agentsare anti-androgenic agents. In an aspect, the one or more therapeuticagents are anti-neoplastic agents.

In an aspect, the one or more therapeutic agents are selected from oneor more antimicrobial compounds, one or more antibacterial compounds,one or more antifungal compounds, or one or more anti-cancer agents, ora combination thereof. In an aspect, a disclosed therapeutic compositioncan comprise one or more anti-cancer agents. In an aspect, the one ormore anti-cancer agents can comprise cisplatin. In an aspect, the one ormore anti-cancer drugs induce apoptosis. In an aspect, a disclosedtherapeutic composition can comprise one or more chemotherapeutic drugs.In an aspect, a disclosed therapeutic composition can comprise one ormore radiosensitizers. In an aspect, a disclosed therapeutic compositioncan comprise a pharmaceutically acceptable carrier.

In an aspect, a disclosed therapeutic composition can comprise (i) oneor more therapeutic agents, (ii) one or more anti-cancer agents, (iii)one or more chemotherapeutic drugs, and/or (iv) one or moreradiosensitizers. In an aspect, a disclosed therapeutic composition cancomprise one or more anti-cancer agents and one or more chemotherapeuticdrugs. In an aspect, a disclosed therapeutic composition can compriseone or more anti-cancer agents and one or more radiosensitizers. In anaspect, a disclosed therapeutic composition can comprise one or morechemotherapeutic agents and one or more radiosensitizers.

In an aspect, disclosed herein is a therapeutic composition comprising aCT20 peptide. In an aspect, a disclosed CT20 peptide can comprise SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and/orSEQ ID NO: 6, or a combination of two or more of SEQ ID NOs: 1-6. Forexample, in an aspect, a disclosed CT20 peptide can beVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a disclosed CT20peptide can be ASLTIWKKMG (SEQ ID NO: 2). In an aspect, a disclosed CT20peptide can be VTIFVAGVLT (SEQ ID NO: 3). In an aspect, a disclosed CT20peptide can be VTIFVAG (SEQ ID NO: 4). In an aspect, a disclosed CT20peptide can be IFVAG (SEQ ID NO: 5). In an aspect, a disclosed CT20peptide can be IWKKMG (SEQ ID NO: 6). In an aspect, a disclosedtherapeutic composition can comprise one or more CT20 peptides, whereinthe one or more CT20 peptides can comprise SEQ ID NO:1, SEQ NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a combinationthereof.

In an aspect, a disclosed therapeutic composition can be administeredsystemically to a subject. In an aspect, the subject can be a mammal. Inan aspect, the mammal can be a primate. In an aspect, the mammal can bea human. In an aspect, the human can be a patient.

In an aspect, a disclosed therapeutic composition can be administered toa subject repeatedly. In an aspect, a disclosed therapeutic compositioncan be administered to the subject at least two times. In an aspect, adisclosed therapeutic composition can be administered to the subject twoor more times. In an aspect, a disclosed therapeutic composition can beadministered at routine or regular intervals. For example, in an aspect,a disclosed therapeutic composition can be administered to the subjectone time per day, or two times per day, or three or more times per day.In an aspect, a disclosed therapeutic composition can be administered tothe subject daily, or one time per week, or two times per week, or threeor more times per week, etc. In an aspect, a disclosed therapeuticcomposition can be administered to the subject weekly, or every otherweek, or every third week, or every fourth week, etc. In an aspect, adisclosed therapeutic composition can be administered to the subjectmonthly, or every other month, or every third month, or every fourthmonth, etc. In an aspect, the repeated administration of a disclosedcomposition occurs over a pre-determined or definite duration of time.In an aspect, the repeated administration of a disclosed compositionoccurs over an indefinite period of time.

In an aspect, following the administration of a disclosed therapeuticcomposition, the cells are sensitized to treatment. In an aspect,following the administration of a disclosed therapeutic composition, asubject can be sensitized to treatment. In an aspect, an increasedsensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, can be measured according to one or more methodsas known in the art for the particular treatment. In an aspect, methodsof measuring sensitivity to a treatment include, but not limited to,cell proliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a disclosed therapeutic composition to the sensitivityof a cell or subject that has not been administered a disclosedtherapeutic composition.

For example, in an aspect, following the administration of a disclosedtherapeutic composition, the cell can be 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, orgreater, more sensitive to treatment than a cell that has not beenadministered a disclosed therapeutic composition. In an aspect,following the administration of a disclosed therapeutic composition, thecell can be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant totreatment than a cell that has not been administered a disclosedtherapeutic composition. The determination of a cell's or a subject'ssensitivity or resistance can be routine in the art and within the skillof an ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than at periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment. For example, in an aspect, thetreatment can modified by changing the dose of a disclosed compositions,the route of administration of a disclosed compositions, the frequencyof administration of a disclosed composition, etc.

Disclosed herein is a cancer therapeutic composition comprising at leastone nanoparticle conjugated with a targeting ligand that is a substratefor a solid tumor-specific cell protein, wherein the nanoparticlefurther comprises one or more therapeutic agents encapsulated in thehydrophobic interior of the nanoparticle. In an aspect, disclosed hereinis a therapeutic composition and one or more anti-cancer drugs.Disclosed herein is a nanoparticle composition and one or moreanti-cancer drugs. In an aspect, the disclosed compositions ornanoparticles can comprise two or more therapeutic agents. Anycombination of one or more drugs that can be encapsulated by thedisclosed nanoparticles (e.g., HBPE) can be used. Examples include, butare not limited, to DNA intercalators (like doxorubicin, cisplatin,carboplatin), topoisomerase inhibitors, microtubule stabilizers (taxol),receptor kinase inhibitors, kinase inhibitors, aromatase inhibitors, andanti-androgens. Also, hydrophobic therapeutics soluble in DMSO, DMF orethanol, with different degrees of hydrophobicity (as shown with theexample of DiI, DiD, and DiR).

Pharmaceutical Compositions

In an aspect, the disclosed subject matter relates to pharmaceuticalcompositions comprising a disclosed composition comprising at least onenanoparticle conjugated with a targeting ligand that is a substrate fora solid tumor-specific cell protein. In an aspect, the disclosedcomposition further comprises an imaging compound and one or moretherapeutic agents encapsulated in the hydrophobic interior of thenanoparticle. In an aspect, the disclosed subject matter relates topharmaceutical compositions comprising a disclosed cancer therapeuticcomposition comprising the disclosed composition. In an aspect, apharmaceutical composition can be provided comprising a therapeuticallyeffective amount of at least one disclosed composition and apharmaceutically acceptable carrier.

Methods Comprising a Disclosed Composition

Methods of Identifying a Solid Tumor Cell Target

Disclosed herein is a method of identifying a solid tumor cell target,comprising: contacting a cell with an effective amount of a compositioncomprising at least one nanoparticle conjugated with a targeting ligandthat is a substrate for a solid tumor-specific cell protein; identifyingone or more nanoparticles bound to the cells by using imaging devices;and optionally, monitoring the solid tumor cell target by repeating thesteps disclosed herein. Optionally, in an aspect, the disclosed methodof identifying a solid tumor cell target can comprise the step oftreating the solid tumor cell by killing or inhibiting its growth.

In an aspect, the solid tumor cell target is a prostate cancer cell. Inan aspect, the prostate cancer cell is castration resistant prostatecancer. In an aspect, the solid tumor cell is a breast cancer cell. Inan aspect, the solid tumor cell is a colon cancer cell. In an aspect,the solid tumor cell is a pancreas cancer cell. In an aspect, the solidtumor cell is a lung cancer cell.

In an aspect, the cells can be individual cells or cells that are on orin a subject. The cells can be individual cells or cells that are on orin a subject. In an aspect, the cells can be in a subject. In an aspect,the cells can be on a surface, which can be inert or can be the surfaceof a subject. In an aspect, the cells are cancer cells or transformedcells. In an aspect, the cancer cells can comprise metastatic cancercells. In an aspect, the cancer cells can comprise mesenchymal stem-likecancer cell. In an aspect, the cancer cell can be a cell from any typeof cancer including, but not limited to, cancer of the head and neckcancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, andcervix. In an aspect, the cancer can be prostate cancer. In an aspect,the prostate cancer can be castration resistant prostate cancer. In anaspect, the cancer can be breast cancer. In an aspect, the cancer can becolorectal cancer. In an aspect, the cancer can be lung cancer. In anaspect, the cancer can be a drug resistant cancer. In an aspect, thecancer cell can be a drug resistant cancer cell. In an aspect, adisclosed therapeutic composition can be administered directly into atumor. In an aspect, a disclosed therapeutic composition can beadministered directly to the cancer cells. In an aspect, a disclosedtherapeutic composition induces death of cancer cells. In an aspect,noncancerous cells do not die.

In an aspect, the nanoparticles are hyberbranched polyester polymericnanoparticles (HBPE-NPs). In an aspect, the nanoparticles are polymericnanoparticles. In an aspect, the nanoparticles can comprise a targetingmoiety. In an aspect, the nanoparticles are conjugated with a targetingligand. In an aspect, the targeting ligand is a folate compound. In anaspect, the targeting ligand is a glutamate compound. In an aspect, thetargeting ligand is a polyglutamated folate compound. In an aspect, thetargeting ligand is glutamate azido urea. In an aspect, the targetingligand is folate azido urea. In an aspect, the targeting ligand isglutamate azido urea. In an aspect, the targeting ligand is abifunctional glutamate-folate hybridized compound. In an aspect, thetargeting ligand is at high density. In an aspect, the targeting ligandis at low density. In an aspect, the targeting ligand is at highvalency. In an aspect, the targeting ligand is at low valency. In anaspect, the targeting ligand is a substrate for a solid tumor-specificcell protein. In an aspect, the solid tumor-specific cell protein isprostate specific membrane antigen (PSMA).

In an aspect, the nanoparticles further comprise an imaging compound. Inaspect, the imaging compound is a PET detectable compound. In an aspect,the PET detectable compound is ⁸⁹Zr. In an aspect, the PET detectablecompound is CU or other PET detectable compounds. In an aspect, thenanoparticles comprise a chelating ligand such as desferrioxamine (DFO).In an aspect, the nanoparticles are polyglutamatedfolate-HBPE-DFO[CT20p]-nanoparticles. In an aspect, the nanoparticlecomprises PEG.

In another aspect, the nanoparticles comprise one or more therapeuticagents that are encapsulated in the hydrophobic interior of thenanoparticle. In an aspect, the one or more therapeutic agents areCT20p. In another aspect, the one or more therapeutic agents are amutant CT20 peptide. In an aspect, the one or more therapeutic agentsare a mitotoxic peptide. In an aspect, the one or more therapeuticagents are anti-metastatic agents. In an aspect, the one or moretherapeutic agents are anti-androgenic agents. In an aspect, the one ormore therapeutic agents are anti-neoplastic agents.

In an aspect, the one or more therapeutic agents are selected from oneor more antimicrobial compounds, one or more antibacterial compounds,one or more antifungal compounds, or one or more anti-cancer agents, ora combination thereof. In an aspect, a disclosed therapeutic compositioncan comprise one or more anti-cancer agents. In an aspect, the one ormore anti-cancer agents can comprise cisplatin. In an aspect, the one ormore anti-cancer drugs induce apoptosis. In an aspect, a disclosedtherapeutic composition can comprise one or more chemotherapeutic drugs.In an aspect, a disclosed therapeutic composition can comprise one ormore radiosensitizers. In an aspect, a disclosed therapeutic compositioncan comprise a pharmaceutically acceptable carrier.

In an aspect, a disclosed therapeutic composition can comprise (i) oneor more therapeutic agents, (ii) one or more anti-cancer agents, (iii)one or more chemotherapeutic drugs, and (iv) one or moreradiosensitizers. In an aspect, a disclosed therapeutic composition cancomprise one or more anti-cancer agents and one or more chemotherapeuticdrugs. In an aspect, a disclosed therapeutic composition can compriseone or more anti-cancer agents and one or more radiosensitizers. In anaspect, a disclosed therapeutic composition can comprise one or morechemotherapeutic agents and one or more radiosensitizers.

In an aspect, disclosed herein is a therapeutic composition, comprisinga CT20 peptide. In an aspect, a disclosed CT20 peptide can comprise SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and/orSEQ ID NO: 6, or a combination of two or more of SEQ ID NOs: 1-6. Forexample, in an aspect, a disclosed CT20 peptide can beVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a disclosed CT20peptide can be ASLTIWKKMG (SEQ ID NO: 2). In an aspect, a disclosed CT20peptide can be VTIFVAGVLT (SEQ ID NO: 3). In an aspect, a disclosed CT20peptide can be VTIFVAG (SEQ ID NO: 4). In an aspect, a disclosed CT20peptide can be IFVAG (SEQ ID NO: 5). In an aspect, a disclosed CT20peptide can be IWKKMG (SEQ ID NO: 6). In an aspect, a disclosedtherapeutic composition can comprise one or more CT20 peptides, whereinthe one or more CT20 peptides can comprise SEQ ID NO:1, SEQ NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a combinationthereof

In an aspect, a method of identifying a solid tumor cell targetcomprising contacting a cell with a disclosed therapeutic compositionthat can induce cell death. In an aspect, the cell death mimicsnecrosis. In an aspect, the cell death occurs independent of endogenousBax activity. In an aspect, the cell death can occur independent ofendogenous caspase activity. In an aspect, the cell death can beresistant to Bcl-2 over-expression.

In an aspect, a method of identifying a solid tumor cell targetcomprising contacting a cell with a disclosed therapeutic compositionthat induces cell death, wherein (i) the cell death mimics necrosis,(ii) the cell death occurs independent of endogenous Bax activity, (iii)the cell death occurs independent of endogenous caspase activity, or(iv) the cell death is resistant to Bcl-2 over-expression, or (v) thecell death exhibits a combination thereof.

In an aspect, a method of identifying a solid tumor cell targetcomprising contacting a cell with a disclosed therapeutic compositionsuch that the disclosed therapeutic composition can be administeredsystemically to a subject. In an aspect, the subject can be a mammal. Inan aspect, the mammal can be a primate. In an aspect, the mammal can bea human. In an aspect, the human can be a patient.

In an aspect, a method of identifying a solid tumor cell targetcomprising contacting a cell with a disclosed therapeutic compositionsuch that the disclosed therapeutic composition can be administered to asubject repeatedly. In an aspect, a disclosed therapeutic compositioncan be administered to the subject at least two times. In an aspect, adisclosed therapeutic composition can be administered to the subject twoor more times. In an aspect, a disclosed therapeutic composition can beadministered at routine or regular intervals. For example, in an aspect,a disclosed therapeutic composition can be administered to the subjectone time per day, or two times per day, or three or more times per day.In an aspect, a disclosed therapeutic composition can be administered tothe subject daily, or one time per week, or two times per week, or threeor more times per week, etc. In an aspect, a disclosed therapeuticcomposition can be administered to the subject weekly, or every otherweek, or every third week, or every fourth week, etc. In an aspect, adisclosed therapeutic composition can be administered to the subjectmonthly, or every other month, or every third month, or every fourthmonth, etc. In an aspect, the repeated administration of a disclosedcomposition occurs over a pre-determined or definite duration of time.In an aspect, the repeated administration of a disclosed compositionoccurs over an indefinite period of time.

In an aspect of a disclosed method of identifying a solid tumor celltarget comprising contacting a cell with a disclosed therapeuticcomposition, the cells are sensitized to treatment following theadministration of a disclosed therapeutic composition. In an aspect, anincreased sensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, can be measured according to one or more methodsas known in the art for the particular treatment. In an aspect, methodsof measuring sensitivity to a treatment include, but not limited to,cell proliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a disclosed therapeutic composition to the sensitivityof a cell or subject that has not been administered a disclosedtherapeutic composition.

For example, in an aspect, following the administration of a disclosedtherapeutic composition, the cell can be 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, orgreater, more sensitive to treatment than a cell that has not beenadministered a disclosed therapeutic composition. In an aspect,following the administration of a disclosed therapeutic composition, thecell can be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant totreatment than a cell that has not been administered a disclosedtherapeutic composition. The determination of a cell's or a subject'ssensitivity or resistance can be routine in the art and within the skillof an ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than at periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment. For example, in an aspect, thetreatment can modified by changing the dose of a disclosed compositions,the route of administration of a disclosed compositions, the frequencyof administration of a disclosed composition, etc.

Disclosed herein is a therapeutic composition and one or moreanti-cancer drugs.

Methods of Treating Prostate Cancer

Disclosed herein are methods of treating prostate cancer. In an aspect,disclosed herein are method for treating prostate cancer, comprisingadministering to a subject diagnosed with prostate cancer an effectiveamount of a nanoparticle composition, comprising at least onenanoparticle conjugated with a targeting ligand that is a substrate fora solid tumor-specific cell protein. In an aspect, the nanoparticlefurther comprises an imaging compound. In an aspect, the nanoparticlehas one or more therapeutic agents encapsulated in the hydrophobicinterior of the nanoparticle. Additional therapeutics and/orradiolabeled compounds can be administered with (either separately,before and/or after, or simultaneously) with the nanoparticles.

In an aspect, the cells can be individual cells or cells that are on orin a subject. The cells can be individual cells or cells that are on orin a subject. In an aspect, the cells can be in a subject. In an aspect,the cells can be on a surface, which can be inert or can be the surfaceof a subject. In an aspect, the cells are cancer cells or transformedcells. In an aspect, the cancer cells can comprise metastatic cancercells. In an aspect, the cancer cells can comprise mesenchymal stem-likecancer cell. In an aspect, the cancer cell can be a cell from any typeof cancer including, but not limited to, cancer of the head and neckcancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, andcervix. In an aspect, the cancer can be prostate cancer. In an aspect,the prostate cancer can be castration resistant prostate cancer. In anaspect, the cancer can be breast cancer. In an aspect, the cancer can becolorectal cancer. In an aspect, the cancer can be lung cancer. In anaspect, the cancer can be a drug resistant cancer. In an aspect, thecancer cell can be a drug resistant cancer cell. In an aspect, adisclosed therapeutic composition can be administered directly into atumor. In an aspect, a disclosed therapeutic composition can beadministered directly to the cancer cells. In an aspect, a disclosedtherapeutic composition induces death of cancer cells. In an aspect,noncancerous cells do not die.

In an aspect, the nanoparticles are hyberbranched polyester polymericnanoparticles (HBPE-NPs). In an aspect, the nanoparticles are polymericnanoparticles. In an aspect, the nanoparticles can comprise a targetingmoiety. In an aspect, the nanoparticles are conjugated with a targetingligand. In an aspect, the targeting ligand is a folate compound. In anaspect, the targeting ligand is a glutamate compound. In an aspect, thetargeting ligand is a polyglutamated folate compound. In an aspect, thetargeting ligand is glutamate azido urea. In an aspect, the targetingligand is folate azido urea. In an aspect, the targeting ligand isglutamate azido urea. In an aspect, the targeting ligand is abifunctional glutamate-folate hybridized compound. In an aspect, thetargeting ligand is at high density. In an aspect, the targeting ligandis at low density. In an aspect, the targeting ligand is at highvalency. In an aspect, the targeting ligand is at low valency. In anaspect, the targeting ligand is a substrate for a solid tumor-specificcell protein. In an aspect, the solid tumor-specific cell protein isprostate specific membrane antigen (PSMA).

In an aspect, the nanoparticles further comprise an imaging compound. Inaspect, the imaging compound is a PET detectable compound. In an aspect,the PET detectable compound is ⁸⁹Zr. In an aspect, the PET detectablecompound is CU or other PET detectable compounds. In an aspect, thenanoparticles comprise a chelating ligand such as desferrioxamine (DFO).In an aspect, the nanoparticles are polyglutamatedfolate-HBPE-DFO[CT20p]-nanoparticles. In an aspect, the nanoparticlecomprises PEG.

In another aspect, the nanoparticles comprise one or more therapeuticagents that are encapsulated in the hydrophobic interior of thenanoparticle. In an aspect, the one or more therapeutic agents areCT20p. In another aspect, the one or more therapeutic agents are amutant CT20 peptide. In an aspect, the one or more therapeutic agentsare a mitotoxic peptide. In an aspect, the one or more therapeuticagents are anti-metastatic agents. In an aspect, the one or moretherapeutic agents are anti-androgenic agents. In an aspect, the one ormore therapeutic agents are anti-neoplastic agents.

In an aspect, the one or more therapeutic agents are selected from oneor more antimicrobial compounds, one or more antibacterial compounds,one or more antifungal compounds, or one or more anti-cancer agents, ora combination thereof. In an aspect, a disclosed therapeutic compositioncan comprise one or more anti-cancer agents. In an aspect, the one ormore anti-cancer agents can comprise cisplatin. In an aspect, the one ormore anti-cancer drugs induce apoptosis. In an aspect, a disclosedtherapeutic composition can comprise one or more chemotherapeutic drugs.In an aspect, a disclosed therapeutic composition can comprise one ormore radiosensitizers. In an aspect, a disclosed therapeutic compositioncan comprise a pharmaceutically acceptable carrier.

In an aspect, a disclosed therapeutic composition can comprise (i) oneor more therapeutic agents, (ii) one or more anti-cancer agents, (iii)one or more chemotherapeutic drugs, and (iv) one or moreradiosensitizers. In an aspect, a disclosed therapeutic composition cancomprise one or more anti-cancer agents and one or more chemotherapeuticdrugs. In an aspect, a disclosed therapeutic composition can compriseone or more anti-cancer agents and one or more radiosensitizers. In anaspect, a disclosed therapeutic composition can comprise one or morechemotherapeutic agents and one or more radiosensitizers.

In an aspect, disclosed herein is a therapeutic composition, comprisinga CT20 peptide. In an aspect, a disclosed CT20 peptide can comprise SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and/orSEQ ID NO: 6, or a combination of two or more of SEQ ID NOs: 1-6. Forexample, in an aspect, a disclosed CT20 peptide can beVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a disclosed CT20peptide can be ASLTIWKKMG (SEQ ID NO: 2). In an aspect, a disclosed CT20peptide can be VTIFVAGVLT (SEQ ID NO: 3). In an aspect, a disclosed CT20peptide can be VTIFVAG (SEQ ID NO: 4). In an aspect, a disclosed CT20peptide can be IFVAG (SEQ ID NO: 5). In an aspect, a disclosed CT20peptide can be IWKKMG (SEQ ID NO: 6). In an aspect, a disclosedtherapeutic composition can comprise one or more CT20 peptides, whereinthe one or more CT20 peptides can comprise SEQ ID NO:1, SEQ NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a combinationthereof.

In an aspect, a method of treating prostate cancer comprisingadministering to a subject a disclosed nanoparticle composition that caninduce cell death. In an aspect, the cell death mimics necrosis. In anaspect, the cell death occurs independent of endogenous Bax activity. Inan aspect, the cell death can occur independent of endogenous caspaseactivity. In an aspect, the cell death can be resistant to Bcl-2over-expression.

In an aspect, a method of treating prostate cancer comprisingadministering to a subject a disclosed nanoparticle composition thatinduces cell death, wherein (i) the cell death mimics necrosis, (ii) thecell death occurs independent of endogenous Bax activity, (iii) the celldeath occurs independent of endogenous caspase activity, or (iv) thecell death is resistant to Bcl-2 over-expression, or (v) the cell deathexhibits a combination thereof.

In an aspect, a method of treating prostate cancer comprisingadministering to a subject a disclosed nanoparticle composition suchthat the disclosed nanoparticle composition can be administeredsystemically to a subject. In an aspect, the subject can be a mammal. Inan aspect, the mammal can be a primate. In an aspect, the mammal can bea human. In an aspect, the human can be a patient.

In an aspect, a method of treating prostate cancer comprisingadministering to a subject a disclosed nanoparticle composition suchthat the disclosed nanoparticle composition can be administered to asubject repeatedly. In an aspect, a disclosed nanoparticle compositioncan be administered to the subject at least two times. In an aspect, adisclosed nanoparticle composition can be administered to the subjecttwo or more times. In an aspect, a disclosed nanoparticle compositioncan be administered at routine or regular intervals. For example, in anaspect, a disclosed nanoparticle composition can be administered to thesubject one time per day, or two times per day, or three or more timesper day. In an aspect, a disclosed nanoparticle composition can beadministered to the subject daily, or one time per week, or two timesper week, or three or more times per week, etc. In an aspect, adisclosed nanoparticle composition can be administered to the subjectweekly, or every other week, or every third week, or every fourth week,etc. In an aspect, a disclosed nanoparticle composition can beadministered to the subject monthly, or every other month, or everythird month, or every fourth month, etc. In an aspect, the repeatedadministration of a disclosed composition occurs over a pre-determinedor definite duration of time. In an aspect, the repeated administrationof a disclosed composition occurs over an indefinite period of time.

In an aspect of a disclosed method of treating prostate cancercomprising administering to a subject a disclosed nanoparticlecomposition, the cells are sensitized to treatment following theadministration of a disclosed nanoparticle composition. In an aspect, anincreased sensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, can be measured according to one or more methodsas known in the art for the particular treatment. In an aspect, methodsof measuring sensitivity to a treatment include, but not limited to,cell proliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a disclosed nanoparticle composition to thesensitivity of a cell or subject that has not been administered adisclosed nanoparticle composition.

For example, in an aspect, following the administration of a disclosednanoparticle composition, the cell can be 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,or greater, more sensitive to treatment than a cell that has not beenadministered a disclosed nanoparticle composition. In an aspect,following the administration of a disclosed nanoparticle composition,the cell can be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant totreatment than a cell that has not been administered a disclosednanoparticle composition. The determination of a cell's or a subject'ssensitivity or resistance can be routine in the art and within the skillof an ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than at periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment. For example, in an aspect, thetreatment can modified by changing the dose of a disclosed compositions,the route of administration of a disclosed compositions, the frequencyof administration of a disclosed composition, etc.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods claimed herein are used andevaluated and are intended to be purely exemplary of the disclosedsubject matter and are not intended to limit the scope of what theinventors regard as their invention. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific aspects which are disclosed and still obtaina like or similar result without departing from the spirit and scope ofthe invention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric.

Example 1

(1) Utilization of a Hyperbranched Polyester (HBPE) Nanoparticle

In one aspect, disclosed are spherically-shaped, highly branched HBPEnanoparticles that encapsulate therapeutic and imaging cargos withintheir hydrophobic nanocavities, without affecting the distribution oftargeting ligands on the nanoparticle's surface. The nanoparticle'ssurface comprise carboxylic acid groups that can be functionalized withtargeting ligands to generate a library of functional targetingnanoparticles with high and low valency. These nanoparticles are easilyfabricated from an aliphatic, biodegradable, hyperbranched polyester(HBPE) polymer (FIG. 2) that displays a defined number of carboxylicfunctional groups. As these carboxylic acid groups are not used forconjugation of therapeutic drugs or imaging agents, they are readilyavailable for conjugation of the targeting ligands at high and lowdensity in such a way that the effect of ligand multivalency and itseffect on tumor targeting can be studied. The HBPE polymer disclosedherein has great advantages over conventional linear polymers (such asPLGA) since: (i) it is highly branched creating unique hydrophobiccavities; (ii) it displays a high number of carboxylic acid groups onits surface for facile labeling; and (iii) its monomer contains anacidic proton that can be easily displaced by a pendant ligand, allowingfurther functionalization of the resulting nanoparticles' cavity tointroduce a chelating ligand for stable encapsulation of radioactiveisotopes for PET imaging. Notice that with current linear polymers, itis difficult to engineer the resulting nanoparticle to achieve theadvantages of the HBPE nanoparticles, as they don't generatewell-defined hydrophilic nanocavities that can be further modifiedchemically to introduce further functionalities. In contrast,dendrimers, although highly branched and containing a high number offunctional groups on the surface, are more difficult to synthesize andto chemically engineer their nanocavities to introduce furtherfunctionalities. Taken together, a main innovative aspect of thecompositions and methods described herein is the use of a HBPE polymerto fabricate a multifunctional theranostic polymeric nanoparticletargeting PSMA via multivalent interaction, while chemically engineeringits nanocavities to incorporate chelating agents for PET imaging andefficiently encapsulating a therapeutic drug.

(2) Using Folates and Glutamate Ligands to Target PSMA

In another aspect, disclosed are the design and screening of folate andglutamate containing ligands to target PSMA. Considering that PSMAutilized polyglutamated folate as its biological ligand and it was shownherein that both glutamic acid- and folic acid-conjugated HBPEnanoparticles target PSMA (FIGS. 5-7), a rationally designed library ofsmall molecules containing both glutamate and folate derivatives weredeveloped to be conjugated onto the HBPE nanoparticles for targetingPSMA. Screening of this compound library generated polyglutamated folatecompounds with higher, more specific binding toward PSMA with minimalbinding to the folate receptor. Conjugation of these ligands was done athigh and low density to study the effect of multivalency on thePSMA-targeting nanoparticle conjugates. The disclosed methods areinnovative, as the methods are directed to the effect of thenanoparticles' ligand density on PSMA targeting, using small moleculeligands (glutamate and folate) scaffolds that were shown to bind toPSMA-expressing cells. Glutamate urea-based small molecules have beenpreviously developed as PSMA inhibitors and PET imaging agents of PSMAexpression in PCa in animal models. These small molecules exhibited goodpharmacokinetic and biodistribution profiles, being able to selectivelyimage PSMA in mice xenografts with high target to non-target tissueratios. Furthermore, glutamate urea-based PSMA inhibitors were alsoconjugated to polymeric nanoparticles to deliver doxorubicin to PSMApositive cells. This previous work clearly demonstrates that smallmolecules can be used to target PSMA; however, the methods disclosedherein are significantly different from these previous investigationssince (1) folic acid, a targeting ligand that has not been tested beforeto target nanoparticles to PSMA was used; and (2) a systematic study onthe effect of a multivalent folate/glutamate ligand presentation on PSMAbinding was performed using a theranostic nanoparticle. As these smallmolecules are more stable and easier to manufacture than monoclonalanti-PSMA antibodies or aptamers, members of the resulting multivalentHBPE nanoparticle library provide a more robust PSMA-targetingnanoplatform to target PCa. Through screening a compound library ofsmall molecules containing folate and glutamate ligands, being displayedon a polymeric nanoparticle at high vs. low density, ananoparticle-small molecule conjugate that specifically binds to PSMAwhile displaying minimal binding to the folate receptor was observed.Even though folate conjugated nanoparticles have been developed totarget the folate receptor, their binding to PSMA has not beeninvestigated.

(3) A Theranostic Approach for Dual Targeting and Imaging

In another aspect, disclosed is the design of a theranostic nanoparticlethat is able to deliver an antiandrogenic drug and a PET imaging tracerto PCa via PSMA targeting. This capability is a unique and translationaladvancement for the treatment of PCa as the PET imaging capabilityallows monitoring of the delivery of the therapeutic nanoparticle. Toendow the nanoparticles with PET imaging capabilities, a method ofgrafting desferrioxamine (DFO) onto the HBPE nanoparticle cavities wasdeveloped. Desferrioxamine (DFO) strongly binds Zr and has been used inthe design of ⁸⁹Zr-PET imaging probes. The HBPE nanoparticle's syntheticprocedure was modified to yield a DFO-grafted HBPE nanoparticle able tochelate ⁸⁹Zr (FIG. 3). Introducing a pendant group with selective⁸⁹Zr-chelating ability into the hydrophobic cavities increased theability of the HBPE nanoparticle to chelate ⁸⁹Zr. These nanoparticleschelate ⁸⁹Zr and encapsulate a hydrophobic drug, while displayingtargeting ligands; thus creating a theranostic nanoparticle thatspecifically bind PSMA. For these studies, abiraterone and MDV-3100 wereselected as therapeutic agents for encapsulation into the PSMA-targetingnanoparticles. Abiraterone and MDV-3100 are PCa drugs, currently onclinical trials for the treatment of PCa and are administered orally.These two drugs work by inhibiting the androgen (testosterone) mediatedpathway that facilitates PCa development. However, clinical assessmentof drug delivery is not currently possible with these drug formulations.Also, enteric uptake efficacy and first pass effects through the liverall decrease the actual availability of the drug to treat PCa.Therefore, disclosed herein is the targeted delivery of abiraterone orMDV-3100 in high concentrations selectively to PCa, which significantlyreduced side effects, while allowing assessment of nanoparticle deliveryvia PET imaging.

The incorporation of ⁸⁹Zr facilitated the assessment of nanoparticlelocalization via PET imaging as this radioisotope is a promisinglong-lived positron emitter for the detection of tumors by PET. The ⁸⁹Zrradionuclide has multiple advantages over the typically used ⁶⁴Curadionuclide such as (1) a half-life of approximately 78.4 h (3.17 days)as opposed to the 12.7 h for the ⁶⁴Cu isotope, (2) a positron yield of22.7% which improves counting statistics when compared to otherradioisotopes, (3) no known toxicity to biological systems, and (4)generation of ⁸⁹Zr is cost effective and highly efficient. Recently,=the use of a ⁸⁹Zr-labeled antibodies to image HER2/neu-positive44 andPSMA-positive45 tumors in vivo was reported and the potential clinicaluse of this radiotracer for localizing and staging these tumors wassuggested. However, a nanoparticle with the capability of chelating ⁸⁹Zrfor PET imaging applications has not been reported. Therefore, disclosedherein are methods of designing, fabricating, and characterizing aDFO-grafted HBPE nanoparticle to chelate ⁸⁹Zr for PET imaging of PSMApositive PCa tumors.

The synthesis and characterization of the first generation HBPEnanoparticles via the solvent diffusion method are disclosed herein. Inthis method, both the hydrophobic polymer and guest molecule to beencapsulated were dissolved in a water-miscible organic solvent (e.g.,DMF or DMSO) and the solution was added drop-wise to a beaker containingwater under constant stirring (FIG. 4). Under these conditions, themiscible solvent rapidly diffused into the water, causing the polymer toself-assemble, forming polymeric nanoparticles encapsulating thehydrophobic molecules within hydrophobic pockets. This process exposedthe hydrophilic segments of the polymer to the aqueous solution,resulting in the formation of carboxyl-functionalized nanoparticles. Thepresence of multiple carboxylic acid groups on the nanoparticle'ssurface enabled the conjugation of multiple targeting ligands, creatinga multivalent targeting nanoparticle. The effect of multivalency on thedetection profile of cancer cells by conjugating folic acid at twodifferent densities (low-folate and high-folate) on iron oxidenanoparticles was studied and their interactions with lung cancer cellsexpressing the folate receptor were studied. Results showed that themultivalent high-folate nanoparticle performed better than its lowfolate counterpart, achieving single cancer cell detection within 15min. Therefore, a high valency polyglutamated folate nanoparticleachieves selective binding to PSMA-expressing PCa cells.

The synthesis and characterization of a first generation HBPE polymer(Mw=42 kDa) that was used to fabricate HBPE nanoparticles (88 nm)encapsulating a variety of hydrophobic molecules, such as near infrareddyes, anti-cancer drugs, and chelated metals was reported. Thenanoparticles' surface carboxylic groups were functionalized with apropargyl group and conjugated with an azide functionalized folic acidligand to yield folate-decorated HBPE nanoparticles [HBPE(DiI)-folate].These nanoparticles delivered Taxol to folate-receptor-expressing cellsresulting in substantial cell internalization and cytotoxicity within 24h. Most recently, it was investigated whether the multivalentHBPE(DiI)-folate nanoparticles can target the PSMA receptor in PCa celllines. In the first set of experiments, various cell lines were exposedwith the HBPE(DiI)-folate nanoparticles and the degree of cellassociated fluorescence was assessed using FACS analysis (FIG. 5).Results showed a significantly large amount of fluorescence associatedwith the CWR 22 prostate cancer cell line, which overexpressed PSMA. Incontrast, the PSMA negative DU145 cell line had a reduced amount of cellassociated fluorescence. Other non-prostatic cancer cell lines that didnot express PSMA had reduced cell associated fluorescence, even whensome of these cells (DU145, HT29, H1650, HeLa and SL-Mel 28) expressedthe folate receptor to some degree. This data indicates that the folatenanoparticles disclosed herein target PSMA in the CWR 22 prostate cancercell line. In additional experiments, the PCa cell lines LNCaP and PC3were used. The LNCaP cell line was ideal for these studies, becausethese cells express the PSMA receptor, but do not express the folatereceptor, while PC3 cells are PSMA and folate negative. The resultsshowed that LNCaP cells incubated with the HBPE(DiI)-folatenanoparticles had a significant amount of fluorescence in the cytoplasmindicating internalization of the HBPE(DiI)-folate nanoparticles (FIG.6). This level of cell-associated fluorescence was not observed whenthese nanoparticles were incubated with the PSMA negative PC3 cells.Most importantly, when LNCaP cells were pre-incubated with PMPA, a knowninhibitor of PSMA, the internalization of the nanoparticles wasdrastically reduced (FIG. 6), indicating that the internalizationoccurred via the PSMA receptor. As neither the LNCaP nor the PC3 cellsexpressed significant amounts of folate receptor, these results indicatethat the HBPE(DiI)-folate nanoparticles were internalized into the LNCaPcell lines via PSMA and can be used to target this receptor in vivo. Inadditional experiments, glutamic acid was conjugated to thenanoparticles to create a multivalent HBPE(DiI)-glutamate nanoparticlesand their internalization in PCa cells was studied. As expected, LNCaPcells internalized a significant amount of these nanoparticles, while nosignificant uptake was observed in PC3 cells or LNCaP cells preincubatedwith PMPA (FIG. 6).

To assess the potential in vivo targeting ability of the nanoparticlepreparations, folate-conjugated HBPE nanoparticles encapsulating thenear infrared dye DiR [HBPE(DiR)-folate nanoparticles] were injectedinto PSMA(+) PC3 tumor-bearing mice. Fluorescence tomographic imagingresults showed a significantly higher accumulation of the Folate-HBPEnanoparticles in the PSMA-transfected PC3 tumor, even 2 h afterinjecting the nanoparticles (FIG. 7). At 24 h, even though sometime-dependent accumulation was observed in the PC3 wild-type (PSMAnegative) tumor through EPR effects, a stronger tumor-associatedfluorescence was observed in the PSMA transfected PC3 tumor, indicatinga higher accumulation of HBPE(DiR)-folate nanoparticles. Taken together,these results strongly indicate that the HBPE nanoparticles disclosedherein target PSMA via the multivalent presentation of folate and/orglutamate ligands to deliver multiple imaging and therapeutic cargos inhigh concentrations to prostate cancer. The generation and screening ofa library of polyglutamated folate compounds conjugated to HBPEnanoparticles resulted in lead nanoparticle conjugates with enhanced andspecific binding to PSMA as opposed to the folate receptor in vivo.

The HBPE nanoparticle synthesis procedure was modified to yield aDFO-grafted HBPE nanoparticle that chelates ⁸⁹Zr. The fabrication of theZr-chelating HBPE nanoparticles starts with the synthesis of aDFO-grafted HBPE polymer. In the synthetic procedure (FIG. 8),diethylmalonate (1) (62.5 mmol), 3-chloroprop-1-ene (62.5 mmol) andpotassium carbonate (312.5 mmol) were taken in acetonitrile and refluxedfor 36 h. In this step, the use of a stoichiometric amount ofchloroprop-1-ene and potassium carbonate as a mild base facilitated therelease of only one acidic proton from 1 and its subsequentmonoalkylation. The resulting monoalkylated product 2 (40.0 mmol), waspurified by flash chromatography and reacted with 4-bromobutyl acetate(48 mmol) in a dry THF solution containing NaH (56 mmol). In this secondstep, the use of NaH as a stronger base and the excess amount of4-bromobutyl acetate ensured the removal of the second acidic proton andthe formation of the dialkylated compound 3. Subsequent deprotection of3 (19.2 mmol) by hydrolysis of the protecting ester groups in an aqueousmethanol solution containing NaOH (67.3 mmol) at 90° C. for 12 h,resulted in the formation of monomer 4 containing a propene group as apendant ligand. Monomer 4 was then polymerized under vacuum usingp-toluenesulfonic acid (100:1 molar ratio) as catalyst. In this step,the rate of polymerization and resulting molecular weight of the polymerwas controlled by varying the temperature and time of vacuumapplication. The resulting propene-grafted polymer 5 was oxidized to anepoxide in order to be reactive to the terminal amine group in DFO.Briefly, 3-chloroperoxybenzoic acid (1.2 mmol) was dissolved into amixture of dry dichloromethane (DCM) and Na₂CO₃ (1.2 mmol) underconstant stirring in an ice bath. To this, the polymer 5 (120 mg),dissolved in dry DCM, was added slowly and then stirred for 72 h. Theoxidized polymer was then precipitated in water to obtain pureepoxy-grafted polymer 6. Finally, polymer 6 (40 mg) was reacted with DFO(0.122 mmol) in a methanol solution containing triethylamine (0.203mmol) under constant stirring, at room temperature for 24 h. The finalDFO-grafted-HBPE 7 polymer was purified by precipitation in water. GPCanalysis of the resulting polymer indicated a molecular weight of 40kDa. DFO-grafted HBPE nanoparticles were synthesized via the solventdiffusion method and nanoparticles of 76±4 nm were obtained (FIGS.9A-9B). These nanoparticles were of similar size to the first generationHBPE disclosed herein (even when containing DFO in the cavities), due toan optimization of the polymerization conditions such as time,temperature and reduced pressure. These nanoparticles were fabricatedusing a Fe³⁺ chelated DFO to facilitate “wrapping” of the DFO around themetal for a better fitting in the nanoparticle's inner cavities. Uponincubation with cold Zr⁴⁺ (in the form of ZrCl₄), the chelated Fe³⁺ waseasily displaced by Zr⁴⁺. This was corroborated by ICP-MS resultsshowing a percent by weight of Zr⁴⁺ to polymer of 0.15% in the finalnanoparticle formulation. These results reveal an easy method to labelthe DFO-HBPE nanoparticles with ⁸⁹Zr for PET studies. The resultsindicate that these nanoparticles encapsulate radioactive ⁸⁹Zr.

The DFO-grafted HBPE nanoparticles were encapsulated with abirateroneand the drug release profiles, as well as the cytotoxicity of theresulting nanoparticles, were evaluated. The amount of encapsulatedabiraterone was estimated following a reported protocol and defined asencapsulation efficiency. Following this procedure, an encapsulationefficiency of 75% was estimated. The nanoparticles were stable in PBS pH7.4 with no leaching of the nanoparticle at this pH (FIG. 10).Similarly, addition of increasing amounts of FBS to these nanoparticlesdid not trigger release of the drug. However, upon incubation at lowerpH (6.0 and 5.0), release of the drug was observed, with a higher rateat pH 5.0. The results indicate that the nanoparticle will release thedrug upon endosomal internalization and subsequent localization withinacidic lysosomes. As it is known to one of ordinary skill in the art,folate-decorated nanoparticle, taken up via the PSMA receptor, isfacilitated by an endosomal mechanism. The cytotoxicity of theabiraterone-loaded nanoparticles to PSMA positive LNCaP cells wascorroborated via cell viability studies and determination of the IC₅₀.The assay indicated an IC₅₀ of 2.55 μM for abiraterone in solution and alower value of 890 nM for the folate-DFO HBPE nanoparticle encapsulatingabiraterone (FIG. 10). These results demonstrate that a lower IC₅₀(greater therapeutic value) for abiratenone is obtained by encapsulatingthe drug within the polymeric nanoparticles targeting PSMA, therefore,facilitating better internalization of the drug. At a concentrationhigher than 2.5 μM for abiraterone alone, only 50% of the cells weredead, whereas more than 75% of the cells (25% viability) were dead atthis concentration with the encapsulated and PSMA targeted drug (FIG.10). Finally, fluorescence microscopy studies of LNCaP and PC3 cellsincubated with folate- and glutamate-HBPE(Abiraterone/DiI), indicated asignificant amount of nanoparticle internalization and cell death inLNCaP cells, but not in PC3 cells or LNCaP cells pre-incubated with aPSMA inhibitor (FIG. 11). Taken together, these studies indicated thatthe polymeric nanoparticles disclosed herein are an ideal nanoplatformto deliver potent drugs to PCa cells, increasing their efficacy.Furthermore, these studies showed that folate and glutamate derivativesselectively target the delivery of these drugs to PCa via PSMA.

A library of nanoparticles that displayed polyglutamated folate ligandsat high and low valency was generated. Members of this library weretested first for PSMA binding in vitro. Then, the most optimal membersof the nanoparticle library were further developed for in vivo deliveryof a PET tracer and an antiandrogenic drug. The following experimentswere conducted:

Experiment 1. Creation and Screening of a Library of Multivalent HBPENanoparticles to Target PSMA

In one aspect, disclosed are methods used to synthesize a rationallydesigned library of glutamate- and folate-containing compounds to beconjugated to the surface of HBPE-DFO nanoparticles. As disclosedherein, nanoparticles with these functionalities bind to PSMA-expressingcells. In addition, in vivo studies indicate that these nanoparticleconjugates localize to PSMA-expressing tumor. However, to identifymolecules that bind more selectively to PSMA and the nanoparticle'soptimal targeting ligand density for optimal binding, a library ofligands containing folic and glutamic acid functionalities in differentorientations were designed, with the goal of identifying a particularligand that specifically bind to PSMA. Furthermore, the effect of thenanoparticle's ligand density on the nanoparticle's PSMA targetingability toward prostate cancer cell lines was investigated. This wasachieved by conjugating the ligands at different densities, creatinghigh valency (HV) and low valency (LV) ligand-nanoparticle conjugates. Aligand which contained both folic and glutamic acid functionalities,when displayed on an HBPE nanoparticle, resulted in a more selectivePSMA-targeting nanoparticle.

Ligand Library Synthesis

To facilitate direct linking of the target molecules to propargylatedHBPE nanoparticles via Huisgen-Sharpless's click chemistry, anazide-functionalized library of compounds was generated. The targetedcollection comprised four scaffolds. Scaffold 1 was represented by agamma/alpha substituted polyglutamic folate moiety, which mimicked theendogenous ligand for PSMA. The number of glutamate units (0-5) wassystematically varied to optimize the length of the ligand for binding.In addition, the polyglutamic unit was made with D-amino acids toprevent cleavage by PSMA due to the enzyme's inherentglutamate-carboxylase and hydrolase activity (FIG. 11). Thegamma-substituted derivatives were synthesized from D-polyglutamates andfolic acid using gamma-selective peptide coupling conditions, while thealpha-substituted analogs were accessed through a similar peptidecoupling with a gamma-protected folate derivative.

Scaffolds 2 and 3 comprised either folate or glutamate azido ureaderived compounds, respectively (FIG. 12). Azido urea glutamates havebeen reported as highly specific PSMA binding inhibitors; however, theirconjugation to nanoparticles for targeting PSMA has not been studied indetail. The effect of ligand length and hydrophobicity weresystematically probed herein by using various amino alkyl azides in boththe folate and glutamate azido urea scaffold sub-classes. The creationof azido urea folate compounds and their use as PSMAinhibitors/targeting ligands has never been reported. The scaffold 2library was readily accessible by reaction of the appropriate alkylazidoamines with the isocyano folate, formed via a Curtius rearrangement of aprotected folic acid derivative. Similarly, protected glutamic acidderivatives were converted to the corresponding isocyanides via aCurtius rearrangement, which then formed the scaffold 3-based seriesupon treatment with the alkylazido amines. This method provided readyaccess to these libraries; however, if there are complications with thesynthesis, then replacing the urea moiety with an amide bond allows theuse of standard peptide coupling chemistry as in scaffold 1.

Scaffold 4 comprised various bi-functional glutamate-folate hybridizedcompounds. The proposed 3 analogs within this category are representedin FIG. 13. While examples of the type of ligands synthesized in thislibrary are provided, it is also recognized that the experience and dataobtained making and testing members of the previous categories resultedin identifying the optimal features in terms of length andhydrophobicity of the ligand spacer. This approach inherently definedthe ideal way to simultaneously present both the folic and glutamic acidfunctionalities on the same binding ligand. Thus, data obtained duringthe course of the program resulted in the creation of ligands slightlydifferent from the ones represented in scaffold 4. Scaffold 4-basedanalogs were synthesized using similar methodology as described for theprevious scaffolds. Protected amino acid derivatives were sequentiallyfunctionalized with glutamate, folate and aminoazide moieties using bothpeptide coupling and Curtius rearrangement reactions. These methodsprovided a highly flexible route to a variety of bi-functional glutamatefolate hybrids. These analogs, and all the previously described scaffoldanalogs, were accessible on 20-50 mg scale and the purity and identityof each compound was determined by NMR, and HPLC/UV/MS analyses. Allanalogs prepared were >98% purity and fully characterized. Additionally,the synthesis of specific analogs of interest on a larger scale wasachievable using these routes.

High Valency (HV) and Low Valency (LV) Nanoparticle Systems

To increase the aqueous solubility of the final HBPE nanoparticleconjugates, the nanoparticle's carboxylic acid groups were firstfunctionalized with polyethylene glycol (PEG). Introduction of PEG ontothe nanoparticles also facilitated reduction of non-specific proteinbinding, facilitating longer blood circulation time and thereforeminimizing liver uptake during animal studies. HBPE (DiI) nanoparticleswere used in these experiments. The fluorescent dye DiI was encapsulatedinto the nanoparticles to facilitate cell culture screening.

To conjugate PEG onto the nanoparticles, polyoxyethylene diamine(diamino PEG, M_(w)=3350, 10 mmol) in PBS buffer (pH=7.4) was added to asuspension of HBPE nanoparticles using conventional water-solubleEDC/NHS (10 mmol, MES buffer pH=6.0) carbodiimide chemistry. The finalreaction mixture was purified using PD-10 column, before quantificationof the number of amino groups on the nanoparticle's surface usingstandard SPDP method. The overall change in surface charge (zetapotential measured using Malvern's zetasizer instrument) furtherconfirmed the successful conjugation of diamino PEG. Next, the resultingpegylated HBPE(DiI) nanoparticles were conjugated with 4-pentynoic acid(10 mmol in DMSO) using carbodiimide chemistry. Briefly, a mixture ofEDC and NHS (10 mmol, MES buffer pH=6.0) was added to the solution of4-pentynoic acid, before incubation with the pegylated HBPE(DiI)nanoparticles (6×10⁻³ mmol in PBS buffer, pH=7.4) for 3 h at roomtemperature. The final reaction mixture was purified using a PD-10column, before assessing the number of propargyl groups on thenanoparticle's surface. The resulting propargylated and pegylatedHBPE(DiI) nanoparticles (6×10⁻³ mmol) were conjugated to thecorresponding members of the azide derivatized compound libraries 1, 2,and 3 via “click” chemistry in 0.1 M bicarbonate buffer, pH 8.5,containing a catalytic amount of CuI (0.01 mmol) in bicarbonate bufferas described. To this solution, an azide-functionalized small molecule(10 mmol in DMSO) was added and incubated overnight at 25° C. Theconjugated HBPE nanoparticles were then purified by dialysis and PD-10column to get rid of the click chemistry reagents, particularly Cut, andcharacterized by particle size analysis, SEM and ICP-MS. Successfulligand conjugation was assessed by UV and FT-IR measurements. Sampleswere stored at 4° C.

The average number of ligands bound to the HBPE nanoparticle wascontrolled by varying the ligand's stoichiometry resulting in HV and LVnanoparticles as described herein. Nanoparticles were categorized as HVwhen the number of ligands per nanoparticle was around 100±20, while aLV was one that had 10±5 ligands per nanoparticle. Briefly, for the HVpreparation, a 10× higher amount of the azide functionalized smallmolecule ligands was used as opposed to the LV preparation (lx). Theproper ratio to guarantee a suitable difference in ligand loading (HVvs. LV) was determined experimentally. Confirmation of the successfulHV- vs. LV-conjugation of small molecule ligands was assessed by UV-Visand fluorescence emission. As members of the rationally designed librarycontained folic acid, the assessment of ligand density on thenanoparticle by these spectrophotometric methods was not a problem. Thesize and degree of polydispersity of the resulting HBPE nanoparticleconjugates were characterized by STEM and DLS. HBPE, HBPE-PEG andHBPE-PEG-Folate nanoparticles were incubated with FBS to estimate theamount of nonspecific protein binding by measuring the increase innanoparticle size by DLS after a 24-h incubation period. Results showedno detectable increase in size of the HBPE-PEG or HBPE-PEG-Folate,whereas the HBPE nanoparticles had an increase of 20 nm in size, due tonon-specific protein absorption. These results indicated that thepresence of folate did not interfere with the ability of PEG to preventnon-specific protein absorption.

Cell Culture Screening

The ability of various members of the HBPE nanoparticle (HV and LV) tobind and internalize into PSMA(+) cells was assessed by confocalmicroscopy and FACS studies. For these studies, a panel of culture cellsthat express different levels of PSMA and FR were used. These studiesassessed the specificity of the HV and LV ligand functionalized HBPEnanoparticles toward PSMA. As positive control, HBPE nanoparticlesconjugated with anti-PSMA antibodies or PSMA aptamers were tested andresults were compared to those obtained with the HV and LVpolyglutamated folate nanoparticles. All cell lines were obtained fromATCC, except the PSMA(+) PC3 cells, which were obtained from MSKCC. Allcells were maintained in accordance to the supplier's protocols in ahumidified incubator at 37° C. under 5% CO₂ atmosphere.

Confocal Laser-Scanning Microscopy

Cells were grown overnight on culture dishes, before treatment. Afterincubation with the nanoparticles, the cells were washed three timeswith 1×PBS, fixed with 5% formalin solution, stained with DAPI(Molecular Probes) for nuclear visualization and finally examined fornanoparticle internalization using a Zeiss LSM 510 confocal microscopeequipped with a 40× objective.

Flow Cytometry

Treated cells were detached and centrifuged at 1000 rpm beforecollecting, washing and suspending the cell pellets in 1×PBS. Theresulting cellular suspensions were examined using a FACSCalibur flowcytometer (BD Biosciences). The specificity of the nanoparticleinternalization via PSMA was assessed by studies using PMPA, a PSMAinhibitor.

Characterization and Testing

All small molecules and intermediates synthesized were characterized byusing common spectroscopic techniques, FTIR, ¹H and ¹³C NMR, HPLC andmass spectroscopy. The nanoparticle conjugates were characterized usingUV-Vis and fluorescence spectroscopic analyses. Size of the conjugatednanoparticles was measured using a Precision detectors Dynamic LightScattering (PD2000 DLSplus) system and by STEM. A successful preparationhad its size nearly unchanged from the starting preparation and wasstable in aqueous buffers.

Data Analysis and Alternatives

The synthetic routes chosen for the syntheses of scaffolds 1-4 wererobust and precedented with no complications beyond the standardoptimization of reaction conditions (time, temperature, solvent, reagentstoichiometry). If unexpected complications arise, the chosen routescontain sufficient flexibility for altering the sequence of reactionsalong the synthetic route. Additionally, the click chemistry used toattach the small molecule library to the nanoparticles would workequally as well if synthetic considerations required that the azide andalkyne moieties to be transposed between substrates. Alternatively,recently developed click chemistry reactions that do not involve the useof Cu catalysis can be used in the case that Cu presents a toxicityproblem during animal studies. However, this is not a problem as thenanoparticles disclosed herein do not bind Cu nonspecifically. ICP-MScharacterization of the nanoparticles was performed to verify theabsence of Cu in the final nanoparticle formulation. The abovenanoparticle experiments resulted in data on optimized reactionconditions (e.g. ratio of nanoparticle to conjugation reagents,incubation times, temperature, etc.). Data was tabulated and the mostoptimal conjugation procedures that result in stable nanoparticleconjugates were chosen for subsequent studies. Only preparations withreproducible syntheses and monodisperse particle size distributions wereused for subsequent experiments.

Statistical Analysis

All optimization experiments were conducted in triplicate. Appropriatecontrols were always included. Means, standard deviations, and graphicswere the primary tools to summarize the data. Correlations wereperformed using the Spearman method. Two-way ANOVA method was used tocompare the differences among different agents and to compare amongdifferent time points within each treatment with a statisticallysignificant difference defined as a P value of less than 0.05.

Experiment 2. Synthesize of ⁸⁹Zr-DFO Grafted Theranostic HBPENanoparticles to Target PSMA

In one aspect, disclosed are procedures for the fabrication of aZr-chelating DFO-grafted HBPE nanoparticle. The developed protocol forthe synthesis of a DFO-grafted HBPE nanoparticle and the subsequentfabrication of a Zr:DFO-grafted HBPE nanoparticles have beenreproducible, yielding a monodispersed nanoparticle preparation of 76±4nm (FIGS. 9A-9B). In this experiment, the synthetic procedure for theDFO-HBPE nanoparticles and the encapsulation protocol for abirateroneand MDV-3100 were optimized. Furthermore, the incorporation of ⁸⁹Zr wasoptimized for potential tracking of the nanoparticle using PET imaging.A DFO-grafted HBPE nanoparticle chelates ⁸⁹Zr as well as encapsulates anantiandrogenic drug (abiraterone or MDV-100) resulting in a theranosticnanoparticle for the treatment of PCa.

Synthesis, Characterization and Optimization of ⁸⁹Zr:DFO-Grafted HBPENanoparticles

In this experiment, the ability of the nanoparticles to chelate ⁸⁹Zr wasstudied, with the goal of fabricating a stable and reproduciblepreparation of ⁸⁹Zr-DFO-grafted HBPE nanoparticle. The radioactive ⁸⁹Zrwas generated and supplied MSKCC. Briefly, zirconium-89 was produced bya (p,n) reaction on natural yttrium-89. A variable-beam energy cyclotron(Ebco Industries Inc., BC, Canada) was used to bombard ⁸⁹Y, resulting inthe displacement of a neutron by a proton, and thus creating ⁸⁹Zr. TheFe³⁺:DFO-grafted HBPE nanoparticles were prepared using the solventdiffusion method as described above. This was accomplished by preparinga DMF (40 μL) solution of Fe-DFO-HBPE (50 mg) and adding it drop-wise(10 μL/drop) to nanopure water (700 μL) with continuous stirring at roomtemperature. The synthesized nanoparticles were purified via dialysis(MWCO 6-8K) against water. Next, optimization experiments were performedto determine the most optimal level of encapsulation for abiraterone andMDV-3100. In preliminary studies, encapsulation efficiency was found forabiraterone of 75% in the HBPE nanoparticles. This was achieved byencapsulating 1 mg of the drug into Fe-DFO-HBPE (50 mg) nanoparticles insuspension. The amount of drug (1-5 mg) was systematically varied inorder to achieve a maximum of drug encapsulation without compromisingnanoparticle stability. All nanoparticle preparations were characterizedby DLS, STEM and ICP-MS to access the amount of incorporated iron. Inaddition, the encapsulation efficiency (EE %) and rate of drug releasein vitro were assessed. In separate experiments, stability tests of thenanoparticle preparations after incubation in serum (FBS) supplementedbuffers were performed by measuring the amount of drug release andincrease in particle size (due to swelling or serum protein binding)upon incubation. All three nanoparticle preparations (1) abiraterone-,(2) MDV-3100- and (3) empty Fe³⁺:DFO-grafted HBPE nanoparticles werethen tested for exchange with radioactive ⁸⁹Zr. The Fe chelated by DFOwithin the nanoparticle was displaced by ⁸⁹Zr as described. In order toremove the iron, an excess EDTA solution was added to the HBPE-DFO-Feand incubated for 30 min at pH 4.5. Subsequently, after thetranschelation was complete, the HBPE-DFO was purified by PD-10 sizeexclusion chromatography. ⁸⁹Zr, in an oxalic acid solution adjusted topH 7.7-8.5, was then added to the purified HBPE-DFO and the reaction wasincubated at room temperature for 1-2 h. After the reaction was completethe HBPE-DFO-⁸⁹Zr was also purified with a PD-10 column (GE Healthcare).

The above experiments resulted in data on optimized reaction conditions(e.g. ratio of metal (Fe, Zr) to DFO-grafted polymers and nanoparticles,amount of drug loaded, incubation times, temperature). Data wastabulated and the most ideal conditions and optimal ratios were chosenfor subsequent studies. Optimized synthesis was scaled up and completerecords of all batch synthesis were kept. Only preparations withreproducible syntheses and particle characteristics were used forsubsequent experiments. While abiraterone and MDV-3100 were chosen asdrugs to be encapsulated, a variety of other drugs can be employed ifdifficulties with this choice were encountered. Some alternativesinclude taxol and doxorubicin. All optimization experiments wereconducted at least in triplicate. Appropriate controls were alwaysincluded.

Statistical Analysis was performed as described in Experiment 1

Experiment 3. In Vivo Assessment of Lead Members of the Nanoparticles inAnimal Models of Prostate Cancer

In this experiment, the expected clinical value of the ⁸⁹Zr-DFO graftedHBPE nanoparticles was evaluated. Their ability to target PCa and todetect bone tumors as a model for bone metastases in a mouse model wasdetermined. PET imaging allowed quantification not only of the PSMAexpression but it also allowed the delivery efficacy of the nanoparticleto the tumor to be judged in the second step. The binding (via StandardUptake Value [SUV] in PET) was correlated with the amount of PSMAexpressed in the tumors. The therapeutic efficacy of PCa drugs wereincreased by encapsulation in ⁸⁹Zr-DFO grafted HBPE nanoparticles whileat the same time allowing monitoring of the drug distribution by PET.

Targeting Subcutaneously Implanted PCa Cells in Mice

The lead members of the multivalent PSMA targeting nanoparticles weretested in vivo with male SCID SHO mice, each bearing a PSMA-positive PC3tumor on one flank, a PC3 wild-type tumor on the other and a LNCaP tumoron the back. This provided a spectrum of PSMA expression to evaluate thein vivo specificity of the HBPE nanoagent as the PSMA expression ishigher in the transfected cell line. First, a biodistribution study wasperformed to obtain information on the tumor uptake of thenanoparticles. To this end, ca. 20 μCi of ⁸⁹Zr-DFO grafted HBPEnanoparticles conjugated to the lead PSMA targeting ligands wereinjected into a cohort of mice (n=3 per time point, tail-vein injection)and the tumors and organs 6, 12, 24, 48, 72 and 96 h were harvestedafter injection. The time point for the following in vivo imagingstudies was based on the biodistribution data. For PET imaging (Focus120, CTI/Siemens, Knoxville, Tenn.), standard uptake values (SUV) weredetermined for the PSMA-positive and -negative tumors. For opticaltomography (FMT2500, VisenMedical, Bedford, Mass.) the concentration ofthe nanoparticles were measured after prior calibration of the systemwith the nanoparticles. Co-registration of PET and FMT was performedusing a specialized imaging cassette that fits into the FMT as well asonto the PET scanner with minimal attenuation and included fiducialmarkers (Visen). Dose finding studies were performed to obtain theminimal required dose for imaging, expected to be at around 125 μCi. Thetumors were harvested for immunohistochemistry to detect PSMA and toconfirm co-localization of the probe via fluorescent microscopy andautoradiography. Western blot analysis was used to quantify the amountof PSMA and for correlation with the imaging data. Based upon thespecific activity of the nanoparticles, the amount of nanoparticleswithin the tumors was estimated. To assess the specific binding of thenanoparticles to PSMA, the following controls were used: (1) HBPEnanoparticle without targeting ligand, (2) PMPA, as a known inhibitor ofPSMA, was co-injected with the nanoparticles and (3) excess(non-conjugated) small molecule ligands were co-injected with thenanoparticles as blocking experiments. In all these controls, thenanoparticles did not bind to the PSMA bearing tumors and the degree ofnon-specific binding was assessed. In addition, control experimentsusing HBPE nanoparticles conjugated with anti PSMA antibodies or PSMAaptamers were tested in vivo and results were compared to those obtainedwith polyglutamated folate nanoparticles.

Targeting Bone Tumors as a Model of Bone Metastasis in Mice

Next, the capabilities of the probes to detect tumors seeded to the boneas a model of cancer metastases were explored. To create bone tumors,the tibiae of mice were exposed and a small hole was drilled through thecortex into the marrow space using a stero-microscope. Once the cavitywas accessed, concentrated PSMA (+) PC3 cells in medium were slowlyinjected until backflow was observed. After flushing of the side toremove back-flushed cells, the drill hole was closed with bone wax andthe skin was closed with sutures to avoid artifacts from metallicstaples on imaging. The mice (n=5) were followed weekly via MR-imagingto detect developing bone tumors. Once tumors were detected, the micewere injected with the corresponding nanoagent as described herein andimaged with PET and FMT. The mice were sacrificed and the number andmean-size of metastases were correlated with the read out obtained byimaging as described herein. Controls comprised mice carrying PSMA (−)PC3 tumors and mice bearing PSMA-positive tumors but injected withcontrol non-targeted HBPE nanoparticles.

In the event that significant uptake with PET is not measured, theexcised tumors can be measured in a well counter, which is moresensitive than PET imaging. If activity is detected with the counter(but not with PET), the ⁸⁹Zr labeling efficacy can be increased byincreasing the ratio of ⁸⁹Zr to DFO-grafted HBPE nanoparticles, enablingmore ⁸⁹Zr to be chelated. Additionally, the dose of nanoparticlesinjected can be increased. For subcutaneous tumors, optical imaging ofthe animals can be conducted after injecting a higher dose of particlesto rule out in vivo de-chelation of the ⁸⁹Zr, in which case a PET signalwill not be acquired but the particles can be detected with FMT asdescribed herein.

The ability of the nanoparticles to carry a therapeutic payload directlyto the targeted tumor was determined and the therapeutic efficacy wascompared with therapy with the free drug. The theranostic HBPE utilizedto improve the delivery of an anti-androgenic (abiraterone or MDV-3100)therapy was studied. Again, the lead nanoparticle preparations wereused. It was first tested in vitro if cell death can be obtained usingthe same cell lines as in the previous experiments by incubating thecells with the theranostic nanoparticles either 24, 48 or 72 h at 3different concentrations. LNCaP cells were treated with a totalconcentration of abiraterone (0.1, 1 and 10 mM dissolved in 10 μL DMSO;Sigma Aldrich) or MDV-3100 (10, 100 or 500 nM; Medivation, SanFrancisco, Calif.). As control LNCaP cells were also treated with eithervehicle (DMSO), empty HBPE nanoparticles (i.e. without the drug aspayload) or the drug delivered freely at the same dosage (i.e. withoutHBPE) only. For the anti-androgen therapy, PC3-PSMA+ cells were used ascontrols since PC3 cells lack the androgen receptor. PC3 wild type andLNCaP-PSMA knock out cells were also utilized as control, both withoutPSMA expression. To these cells, the HBPE nanoparticles were nottargeted specifically due to the lack of PSMA. The amount ofnanoparticles taken up into the cells and the proportion of dead ordying cells, respectively, were determined via FACS for all groups(using 7AAD as a marker of apoptosis). Additionally, fluorescencemicroscopy of the nanoparticle treated cells was performed. Thepercentage of apoptotic cells in each group was compared. In vivostudies were conducted subsequent to the in vitro studies. To this end,groups of mice with both LNCaP wild type and LNCaP-PSMA-negative tumorson each flank were used. The mice (n=5 per group) were treated witheither HBPE/abiraterone or HBPE/MDV-3100 nanoparticles on 3 consecutivedays. Three different dosages were tested: 0.1, 0.5 or 1.0 mmol/kgabiraterone and 10, 25 or 50 mg/kg MDV-3100 injected iv on 3 consecutivedays as described. Control mice received empty (HBPE alone) vehicle orthe free drugs at the same dosage without nanoparticle carrier. Tumorgrowth was monitored by measuring the tumor size. At the same time,combined optical and PET imaging was performed to monitor the targetingof the nanoparticles to the tumors as described herein. Imaging wasperformed 24 h after the first and the last dose; and SUV values wereobtained from the tumors. No imaging was performed in mice not receivingparticle preparations. In addition, blood was collected for weekly PSAmeasurements (with a commercially available ELISA), using the valueprior to therapy as a baseline. The tumors were followed for 6 weeks oruntil reaching 1.5 cm in size (whichever comes first). The tumors wereharvested for immunohistochemistry (using J591 to identify PSMA) andalso qrt-PCR and quantitative Western Blot for PSMA levels in the tumorto correlate with response to the targeted therapy. The study wasrepeated with the best dosing, using lung colonies as described herein.The size, weight, and growth dynamic of the tumors were correlated whereapplicable with the SUV value (indicating the amount of targeting HBPEnanoparticle) as well as with the dose of the drug applied with theparticles, the amount of PSMA within the tumors and the serum PSAvalues.

With the completion of the third experiment, the following wereobserved: (1) one of the lead polyglutamated folate HBPE nanoparticlesdelivers therapy via targeting, (2) the therapy delivery was improvedover its conventional form, and (3) the therapy efficacy of thenanoparticles were established for 2 different drugs.

In the unlikely event that there is no response to the therapy, eitherin vitro or in vivo, is observed with the chosen doses, the dose can beincreased gradually. If the dose is too high (i.e. toxic), less dose canbe given over more days. If this does not result in the expected effect,go back to the library and utilize the second best carrier with theexpectation that it will fare better in vivo. An increase of PSMAexpression upon androgen deprivation has been documented in theliterature. It is, therefore, possible that increased binding efficacyof the nanoparticles can be observed after the last dose of therapy. Ifthis is the case, this effect can be utilized by first treating withantiandrogens to increase the PSMA expression, followed byHBPE/etoposite nanoparticles.

For an expected difference in means of at least 75% and a power of 95%,a sample size of 3 was calculated. To account for biologicalvariability, a sample size of n=5 mice per group were used for all invivo experiments. All in vitro experiments were done in triplicate, andin vivo experiments were repeated for reproducibility. Help with allstatistical analysis were obtained from the Biostatistics Core of MSKCC.Support from this core included the determination of overallexperimental designs, hypothesis generation, interim analysis, datamanagement, power, quality control of research data, and finalstatistical analysis. Analyses of data were descriptive in nature.Means, standard deviations, and graphics were the primary tools tosummarize the resulting data. Correlation was performed using theSpearman correlation method. Two-way ANOVA method was also used tocompare the differences among of different agents and to compare amongdifferent time points within each treatment with a statisticallysignificant difference defined as a P value of less than 0.05. For theacute biodistribution studies, a Student's t test was performed usingGraphPad PRISM (San Diego, Calif.). Differences at the 95% confidencelevel (p<0.05) were considered significant.

Example 2

Therapeutic peptides, with cancer cell specific activity, are apromising treatment option for mCRPC. CT20p, a mitotoxic peptide,disclosed herein, targets cancer-specific differences in mitochondrialphysiology. CT20p is a promising anti-metastatic agent because it causesdetachment-induced cell death; however, to develop the clinical use ofCT20p for mCRPC, there are challenges that need to be met, such as lowstability in serum. Disclosed herein is a targeted molecularnanotheranostic (dual therapy and diagnostic) platform that deliversCT20p in high concentrations to PCa and has the capacity for imagingpeptide efficacy in murine models of PCa. To deliver CT20p to PCa, thepeptide was encapsulated within hyperbranched polyester nanoparticles(HBPE-NPs) that were functionalized with polyglutamated folates, thenatural ligand for a PCa-specific cell surface protein, PSMA. PSMA ishighly expressed in PCa tumors and metastatic lesions but not normalprostate. To endow the NPs with imaging capabilities, the polymer wasmodified to graft desferrioxamine (DFO), a chelating ligand for stableencapsulation of a ⁸⁹Zr-PET imaging probe. PSMA-targeted HBPE[CT20p]NPs,co-encapsulated with ⁸⁹Zr, yield a powerful therapeutic platform toreduce PCa growth and metastatic spread, while enabling assessment ofparticle biodistribution. In one aspect, disclosed are methods for thesynthesis of HBPE-DFO[CT20p]-NPs, in which the HBPE-DFO[CT20p]-NPs wasoptimized to obtain effective chelation of ⁸⁹Zr, pegylation and CT20ploading (Experiment 1). In another aspect, a series of polyglutamatedfolate-HBPE-DFO[CT20p]-NPs were synthesized and tested to target PCacells via PSMA (Experiment 2). PET imaging, using murine models of PCa,was used to assess delivery and efficacy of CT20p and pharmacokinetics.The clinical value of the HBPE-DFO[CT20p]-NPs disclosed herein wasinvestigated in murine models of PCa, using mice that were intact orcastrated, and in models of lymph node and bone metastasis (Experiment3). PSMA-targeted, HBPE-DFO[CT20p]-NPs (without ⁸⁹Zr) can be directlyused for the treatment of mCRPC without the side effects associated withcurrent therapies, while the theranostic version (with ⁸⁹Zr) providesthe pre-clinical data to advance the use of PET imaging for monitoringfast growing prostate tumors and treatment outcomes.

A targeted, multifunctional nanoparticle platform incorporating atherapeutic peptide is disclosed herein as a treatment approach forcastration resistant prostate cancer and metastatic disease. Theapproach involves engineering the nanoparticle platform to encapsulatethe therapeutic peptide and chelate ⁸⁹Zr for dual treatment and PETimaging in prostate cancer mouse models. Positive outcomes were measuredin the capacity to monitor the disease-specific accumulation ofnanoparticles in tumors and stimulate tumor regression.

Prostate cancer (PCa) is a leading cause of cancer deaths in men.Current therapies, such as androgen deprivation treatment (ADT), areinitially effective but have severe side effects, including impotenceand incontinence. Over time, nearly all men develop progressive diseaseor castration-resistant prostate cancer (CRPC), which has a poorprognosis, especially if the cancer has spread. CRPC patients with bonemetastasis have survival rates of less than 2 years and most treatmentapproaches for metastatic CRPC only extend life by a few months. Hencethere is a need for more effective anti-metastatic CRPC therapies.Peptides therapeutics, specifically those designed to impairmitochondrial energy-providing functions, are promising treatmentoptions for metastatic disease that significantly improve the quality oflife and survival of patients with CRPC. Recently, CT20p, a mitotoxicpeptide that targets cancer-specific differences in mitochondrialphysiology, disrupting cell adhesion and causing detachment-induced celldeath was discovered. CT20p has the potential to impede cancer cellinvasiveness, making the peptide a promising agent for inhibitingmetastasis. However, in order to develop the clinical use of CT20p forlife-threatening cancers like CRPC, there are challenges that need to bemet, including low stability of the peptide in serum, degradation byproteases, and lack of peptide monitoring during pre-clinicalbio-distribution studies. New platform technologies that allow for theconcentration and monitoring of therapeutic peptides to areas of diseaseare urgently needed. Nanoparticle (NP)-based technologies are effective,because nanoparticles (NPs) stably incorporate and protect peptides,like CT20p, from proteases, while enhancing cellular uptake via the useof targeting ligands. While the hydrophobic nature of CT20p limits itsdirect use in cell culture and animal studies, this facilitates theencapsulation of the peptide in hyperbranched polyester NPs (HBPE-NPs)that can also incorporate imaging agents within the polymer matrix. SuchNP formulations allow for the monitoring of particle biodistribution,using highly sensitive imaging technologies such as PET (positronemission tomography), and have the potential of translational use as anon-invasive method for monitoring patient outcomes. In addition, theoptimization of ligands on the surface of HBPE-NPs increases targetingof NPs, improving concentration in tumors and metastatic sites bearingtargeted receptors. With the goal of developing a platform technologyfor the delivery and monitoring of a therapeutic peptide for thetreatment of CRPC, second generation HBPE-NPs were fabricated thatincorporate CT20p and ⁸⁹Zr for assessment by PET imaging. In preliminarystudies, HBPE[CT20p]NPs caused PCa tumor regression in treated mice withno detectable toxicity to normal tissue. Targeted HBPE[CT20p]NPs,co-encapsulated with ⁸⁹Zr, yielded a robust therapeutic platform toreduce PCa growth and metastatic spread, while enabling assessment ofparticle biodistribution. To this end, the hyperbranched polymer wasgrafted with desferrioxamine (DFO), a ligand that chelates ⁸⁹Zr, along-lived positron emitting radioisotope (half-live of 3 days). Totarget PCa cells, the prostate specific membrane antigen (PSMA), amembrane-bound receptor that correlates with the severity of PCa and isexpressed in metastatic lesions but not normal cells was utilized. Inpreliminary results, PSMA was an effective targeting receptor. Asystematic optimization of the NP preparation was performed tofacilitate the incorporation of ⁸⁹Zr within the NPs nanocavities and tooptimize CT20p loading. To enhance ligand presentation on theHPBE(CT20p)NPs for targeting to PSMA, a series of polyglutamated folatepeptides, since polyglutamate folate is a natural ligand for PSMA, wereconjugated onto the NPs. The following experiments were designed todevelop the best HBPE-NP conjugates for targeted imaging and peptidedelivery to treat PCa.

Experiment 1. Synthesis and Optimization of HBPE-DFO[CT20p]-NPs

The developed protocol for the synthesis of a Fe(III)-DFO-graftedHBPE-NPs encapsulating CT20p is highly reproducible, yieldingmonodispersed NP preparations that average 80 nm in size (FIG. 17B).Recent reports indicated that a NP size of less than a 100 nm indiameter is the most optimal for PCa tumor targeting using PLGA/PLApolymeric NPs. However, other parameters such as amount of PEG on theNP's surface, surface charge (zeta potential) and ligand density play akey role in the stability, targeting ability and pharmacokinetics of theNP formulation. The polymeric NP synthesis protocol was optimized takinginto consideration these parameters. Furthermore, the encapsulation ofthe mitotoxic peptide, CT20p, was optimized and ⁸⁹Zr forbio-distribution studies using PET (Experiment 2) was incorporated. Itwas determined that a stable and monodispersed DFO-grafted HBPE-NPformulation that is optimally PEGylated can be developed with theability of encapsulating CT20p and chelating ⁸⁹Zr. The grafting of DFOwas optimized to obtain effective chelation of ⁸⁹Zr without compromisingPEGylation, CT20p loading, NPs stability, particle size, orpolydispersity. In vitro peptide stability and toxicity studies werealso performed.

Optimization of PEG Conjugation on HBPE NPs

A 12 carbon PEG (carboxy-PEG12-amine) was used to modify the HBPE-NPsherein, and it was established herein that pegylation did not reducecellular uptake of HBPE-NPs loaded with DiR (by flow cytometry). In thisexperiment, PEG length was optimized by conjugatingcarboxy-PEG_(n)-amine of different lengths (n=12, 24, 48, etc.) and thestability of the NPs was assessed in buffer and in serum. To conjugatethe PEG molecules onto the NPs, carboxy-PEG_(n)-amine (Thermo, 10 mmol)in PBS buffer was added to the DFO-HBPE-NPs using conventionalwater-soluble EDC/NHS (10 mmol, MES buffer pH=6.0) carbodiimidechemistry. Folic acid was conjugated to some of these NPs for use ascontrols in Experiment 2. The final reaction mixture was purified usinga PD-10 column. All NP preparations were characterized by DLS, STEM andFTIR to access their polydispersity, and degree of PEG conjugation.Stability tests of the NP preparations after incubation in serum (FBS)supplemented buffers were performed by measuring the increase inparticle size (swelling due to binding of serum proteins). In addition,binding studies with PCa cells (Table 1) were performed to determine howthe different PEG units affect the binding of NPs to cells. The mostoptimal PEG-modified NP preparations were those that resulted in minimalserum protein adsorption, enhanced binding to PSMA(+) cells and improvedcirculation in vivo (See Experiment 2: PK studies).

Optimization of 89Zr:DFO grafted (CT20p) HBPE NPs

The ability of the NPs to chelate ⁸⁹Zr was optimized in order tofabricate stable and reproducible preparations of ⁸⁹Zr-DFO-grafted(CT20p) HBPE-NPs. Radioactive ⁸⁹Zr was generated and tested at the MSKCC(see Letter of Support). Briefly, zirconium-89 was produced by a (p,n)reaction on natural yttrium-89. A variable-beam energy cyclotron (EbcoIndustries Inc., BC, Canada) was used to bombard 89Y, resulting in thedisplacement of a neutron by a proton, and thus creating ⁸⁹Zr. The Fe³⁺DFO-grafted (CT20p) HBPE-NPs, in which Fe was replaced by Zr, wereprepared using the solvent diffusion method as explained herein. A DMF(40 mL) solution of Fe-DFO-HBPE (50 mg) was prepared, adding itdrop-wise to nanopure water (700 mL). CT20p(Ac-VTIFVAGVLTASLTIWKKMG-NH₂) (SEQ ID. NO. 7) and two control peptideswith irrelevant sequences were commercially synthesized at >98% purity(Biopeptide Inc). Peptides were added to the solution as describedherein. The synthesized NPs were purified via dialysis (MWCO 6-8K)against water. Immediately before the murine PET studies (Experiment 2),the Fe chelated by DFO within the NP was displaced by ⁸⁹Zr as describedherein. In order to remove the iron, an excess EDTA solution was addedto the HBPE(CT20p)-DFO-Fe. Subsequently, after the transchelation wascomplete, the HBPE(CT20p)-DFO was purified by PD-10 size exclusionchromatography. ⁸⁹Zr, in an oxalic acid solution, was then added to thepurified HBPE(CT20p)DFO. After the reaction was complete, theHBPE(CT20p)-DFO-⁸⁹Zr was also purified with a PD-10 column (GEHealthcare). The ⁸⁹Zr labeling efficiency of the Fe(III)-DFO-HBPE(CT20p)NPs was accessed by Instant Thin Layer Chromatography (ITLC) and PET b.The stability of encapsulation of the peptides in all NP preparationswas assessed by measuring the rate of release in buffer at physiologicalpH (˜pH 7.4) or at acidic pH (˜pH 4-5) using a microdialysis device asperformed herein. Only preparations with reproducible synthesis andmonodispersed particle size distributions were used for subsequentexperiments.

In Vitro Peptide Stability Assays

In order to perform the PK studies in Experiment 2, the profile of CT20pand control peptides, and any fragments that resulted from these, wasdetermined by mass spectrometry (MS). Peptides were analyzed by LC-MRM(liquid chromatography multiple reaction monitoring mass spectrometry)to define assay parameters. Then a fragment ion spectrum was collectedusing MS/MS and the collision energy was optimized for each fragment.This established the peptide profile. Next, peptides alone orencapsulated in HBPE-NPs, as described herein, were incubated insolutions spiked with mouse and human serum at multiple concentrations(10-10,000 ng/ml), at 4° and 37° C., from 0-48 hours. After incubation,serum proteins were precipitated by methods (e.g. acetonitrile,trichloroacetic acid) that were optimized to ensure maximal peptiderecovery. Recovered solutions were analyzed using LC/MS/MS, as describedherein, to determine peptide stability in serum alone as compared toencapsulation in HBPE-NPs.

In Vitro Toxicity Studies

Previous reports showed that HBPE-NPs are non-toxic to cells in culture.However, since the HBPE-NPs disclosed herein contain Zr-DFO, toxicitystudies were performed using a broad dose range with hepatocytes(HEP10), macrophages (THP-1, RAW 264.7) and fibroblasts (3T3). Celldeath was accessed by Sytox (dead cell stain) and apoptosis using theViolet Ratiometric Membrane Asymmetry Probe (Invitrogen) as shownherein. To demonstrate that the cancer cell-specific killing action ofthe CT20p loaded in PEGylated, DFO-HBPE-NPs or folate-DFO-HBPE-NPs wasunchanged, a panel of PCa cells (Table 1) and non-tumorigenic cells(normal prostate epithelial cells, PCS-440-010) were used and cell deathwas assessed as described herein. Clonogenic assays with a broad doserange were also performed to generate cell survival curves (Rafehi, H.,et al., Clonogenic assay: adherent cells. Journal of visualizedexperiments: JoVE (2011)).

The above experiments resulted in data on optimized reaction conditions,degree of PEGylation and non-interference with targeting ligands,peptide loading, DFO-grafted HBPE-NP yields and peptide stability inserum as well as in vitro toxicity. The peptide profile by LC/MS/MS wasalso determined for PK studies in Experiment 2. Data was tabulated andonly preparations with reproducible syntheses, optimal Zr chelation,CT20p loading, peptide and particle stability, PEGylation and minimaloff-target toxicity were used for subsequent experiments. The dataindicates that functional HBPE-NPs that are inherently non-toxic aregenerated, however NPs that fail STEM or TEM, have inadequate peptideloading or stability or display toxicity in normal cells are detectedand protocols are improved to ensure optimal fabrication. In the eventthat larger NPs (>200 nms) are obtained, the amount of polymer used issystematically reduced. Likewise, when the amount of encapsulatedpeptide is low, the amount of cargo is increased. If the NP preparationis unstable in serum or increases in size due to protein binding, longerPEG linkers are used.

Means, standard deviations, tables and graphics were the primary toolsused to summarize the data. Correlations were performed using theSpearman method. Two-way ANOVA method was used to compare thedifferences among different agents and to compare among different timepoints within each treatment with a statistically significant differencedefined as a P value of less than 0.05.

Experiment 2. Synthesis and Characterization of a Series ofPolyglutamated Folate—HBPE-DFO[CT20p]-NPs to Target PCa Cells Via PSMA

Polyglutamated folate peptide derivatives were conjugated to theHBPE-DFO[CT20p]-NPs to enable binding and internalization intoPSMA-expressing PCa cells. The best NP conjugate that targets PSMA wasidentified, using PSMA(+) and (−) PCa cells. Pharmacokinetic (PK) and invivo toxicity studies were performed along with assessment ofbio-distribution of polyglutamated folate-HBPE-⁸⁹Zr-DFO[CT20p]-NPs byPET imaging using murine models of PCa.

In this experiment, using the disclosed ⁸⁹Zr:DFO-HBPE-NPs-encapsulatingCT20p (from Experiment 1), targeting capabilities were added in the formof polyglutamated folate ligands. This resulted in a targeted NPformulation to bind PSMA. The approach involved the synthesis of afolate ligand conjugated with various glutamate residues via peptidebonds (FIG. 23). The resulting polyglutamated folate ligands, which arepeptides, were synthesized using standard solid phase peptide synthesisprocedures with a cysteine residue at the C-terminus to facilitateconjugation to the NPs, using an established protocol (e.g. maleimide,SPDP linkers). The folate was attached via peptide synthesis couplingand β-alanine was used as a linker between the folate and thepolyglutamate chain to minimize hydrolysis due to the folate hydroxylaseenzymatic activity of PSMA. The polyglutamic acid peptide was made withD-amino acids, making the ligand more resistant to PSMA glutamatecarboxypeptidase activity. As the endogenous substrate of PSMA is aγ-polyglutamated folate, the polyglutamic acid portion of the peptidewas synthesized via γ-peptide coupling. As controls, α-polyglutamatedfolates peptides were generated and tested for binding to PSMA. Thisgenerated multiple polyglutamated folate ligands [Folate-(Glu)n-Cys]that conjugated to HBPE-DFO[CT20p]-NPs to generate a series ofpolyglutamated folate-HBPE-NPs of various lengths (FIG. 23). All NPformulations were characterized for degree of ligand conjugation, size,shape and CT20p loading. For screening purposes, a panel of PCa cellsthat express different levels of PSMA and folate receptor (FR) was used(Table 1) (Hattori, Y., et al., Folate-linked nanoparticle-mediatedsuicide gene therapy in human prostate cancer and nasopharyngeal cancerwith herpes simplex virus thymidine kinase. Cancer Gene Ther 12, 796-809(2005); Xu, L., et al., Tumor-targeted p53-gene therapy enhances theefficacy of conventional chemo/radiotherapy. J Control Release 74,115-128 (2001)).

In vivo toxicity and PK studies were performed with the lead compounds.To demonstrate the PSMA-specific targeting of polyglutamated folate⁸⁹Zr:DFO-HBPE(CT20p)-NPs to PCa tumors, biodistribution studies wereconducted in mouse models of PCa using PET imaging. The binding (viaStandard Uptake Value [SUV] in PET) was correlated with the amount ofPSMA expressed in the tumors. PET imaging allowed quantification notonly of the PSMA expression at tumors but it also allowed the deliveryefficacy of the NP localizing to the tumors to be judged. It wasdetermined that ⁸⁹Zr:DFO HPBE[CT20p]-NPs that are PEGylated andfunctionalized with polyglutamated folate ligands specifically targetPSMA on PCa.

Peptide Synthesis

A total of 10 peptides were synthesized by Fmoc solid phase peptidechemistry. Five peptides were synthesized following a γ-peptidesynthesis approach and the 5 others were synthesized by α-peptidesynthesis (FIG. 23). Peptides had a C-terminal cysteine group, forcrosslinking to the DFO-HBPE-(CT20p)-NPs using N-succinimidyl3-(2-pyridyldithio) propionate (SPDP, Thermo Scientific) as linker. SPDPis a heterobifunctional linker that contains two reactive moieties, anN-hydroxysuccinimide (NHS) ester that reacts with primary amines and anpyridinyldisulfide that reacts with a thiol group, yielding a disulfidelinker that connects the polyglutamated folate peptide to the HBPE-NPs(Josephson, L., et al., High-efficiency intracellular magnetic labelingwith novel superparamagnetic-Tat peptide conjugates. Bioconjugatechemistry 10, 186-191 (1999); Perez, J. M., et al., Magnetic relaxationswitches capable of sensing molecular interactions. Nature biotechnology20, 816-820 (2002)). An advantage of using this chemistry is that thepeptide is linked to the NP by a disulfide bond that is highly stable inaqueous solutions and physiological conditions, while the disulfide bondis sensitive to reducing agents (e.g. DTT), facilitating cleavage foreasy characterization (Perez, J. M., et al., Use of magneticnanoparticles as nanosensors to probe for molecular interactions.Chembiochem: a European journal of chemical biology 5, 261-264 (2004);Perez, J. M., et al., DNA-based magnetic nanoparticle assembly acts as amagnetic relaxation nanoswitch allowing screening of DNA-cleavingagents. Journal of the American Chemical Society 124, 2856-2857 (2002);Perez, J. M., et al., Viral-induced self-assembly of magneticnanoparticles allows the detection of viral particles in biologicalmedia. Journal of the American Chemical Society 125, 10192-10193(2003)). Using this chemistry, the number of glutamate units (0-5) weresystematically varied to optimize the length of the ligand for binding.In addition, the polyglutamic unit was made with D-amino acids toprevent cleavage due to PSMA's inherent glutamate-carboxylase andhydrolase activity (FIG. 23). The γ-substituted derivatives weresynthesized from D-polyglutamates and folic acid using γ-selectivepeptide coupling conditions, while the α-substituted analogs wereaccessed through a similar peptide coupling with a γ-protected folatederivative.

HBPE Conjugation

To increase the aqueous solubility of the final HBPE(DFO)-NP conjugates,the NP's carboxylic acid groups were first functionalized withpolyethylene glycol (PEG) as described in Experiment 1. The overallchange in surface charge (zeta potential measured using Malvern'szetasizer instrument) further confirmed the successful conjugation ofdiamino PEG. Next, the resulting amino-PEG-HBPE-NPs was conjugated withSPDP (10 mmol in DMSO) as described. Briefly, the amino-PEG-HBPE-NPs wasincubated overnight with SPDP (75 μM) and excess was removed using aPD-10 column. Then, the SPDP-activated HBPE-NPs (containing apyridinyldisulfide reactive group) were incubated overnight with thepolyglutamated folate peptides that contain C-terminal cysteine (thiol)groups. The conjugated HBPE NPs were purified by dialysis and PD-10column. Successful ligand conjugation was assessed by UV and FTIRmeasurements. The average number of ligands bound to the HBPE-NPs wascontrolled by varying the ligand's stoichiometry resulting in amultivalent ligand display of peptides on the HBPE surface. Preliminarystudies were done by incubating HBPE-NPs, PEG-HBPE-NPs orfolate-PEG-HBPE-NPs with FBS to estimate the amount of nonspecificprotein binding by measuring the increase in NP size by DLS after a 24-hincubation period. Results showed no detectable increase in size of thePEG-HBPE-NPs or PEG-Folate-HBPE-NPs after FBS incubation, whereas thenonpegylated HBPE-NPs had an increase of 20 nm in size, presumably dueto non-specific protein absorption. These results indicate that folatedoes not interfere with the ability of PEG to prevent non-specificprotein absorption.

In Vitro Targeting and Efficacy Studies

To examine targeted cancer cell killing by polyglutamated folateconjugated DFO-HBPE (CT20p)-NPs, PCa cell lines from Table 1 were usedand viability and survival assays described in Experiment 1 wereperformed. Control NPs included PEGylated, (1) folateDFO-HBPE(CT20p)-NPs (from Experiment 1), (2) polyglutamated folateDFO-HBPE-NPs without CT20p, (3) non-targeted DFO-HBPE(CT20p)-NPs, (4)polyglutamated folate DFO-HBPE-NPs with control peptides. To furtherassess the lack of toxicity of the PSMA-targeting DFO-HBPE-NPs, a panelof non-tumorigenic cell lines such as normal prostate epithelial cells(e.g. ATCC, PCS-440-010), and hepatic cells (e.g. ATCC, CRL-11233), wereincubated with the NPs and cytotoxicity was assessed as describedherein.

In Vivo Toxicity

To examine in vivo toxicity, a subchronic intravenous toxicity assay wasperformed. Groups of male SCID mice (no tumors) were treated weekly withintravenous injections of PEGylated, polyglutamate folate conjugatedHBPE-DFO[CT20p]-NPs for 12-13 weeks at doses ranging from 2-20mg/kg/dose. Mice were observed daily and blood was routinely collectedfrom each mouse for standard clinical chemistry analysis of kidney andliver function (IDEXX-Radil). At experimental endpoints, tissues fromliver, kidneys, spleen and lungs were mounted for histologicalexamination using H & E staining to detect any treatment effect. Serumand urine were collected for detection of anti-PEGIgM (ELISA) and freehemoglobin was measured in the urine to assess if treatment causeshemolysis.

In Vivo PK Studies

PK studies were performed with SCID mice treated with PEGylated,polyglutamate folate conjugated HBPE-DFO[CT20p]-NPs or controls for 24hours. Blood samples were collected from groups of mice after treatments(e.g. 0, 0.5, 1, 2, 4, 6, 8, 12, 24 h), plasma recovered, plasmaproteins precipitated and supernatants subjected to LC/MS/MS analysis(as described in Experiment 1). This data was analyzed followingstandard PK parameters using non-compartment analysis to determine AUC(area under the concentration time curve), CL (total body clearance),MRT (mean residence time), the distribution half-life (T_(1/2α)) andelimination half-life (T1/_(2β), C_(max) (the peak concentration) andt_(max) (the time to reach peak concentration. Furthermore, to assessthe long circulation time of the polyglutamated folate NPs in vivo, the⁸⁹Zr:DFO-grafted (CT20p) HBPE-NPs from Experiment 1 was injected to SCIDmice and blood samples were collected at various time points and samplesprocessed as described herein. The level of ⁸⁹Zr-radioactivity in theblood supernatant was assessed by scintillation counting.

In Vivo Targeting Subcutaneously Implanted PCa Cells in Mice

The most optimal multivalent PSMA targeting NPs were tested in vivo withmale

SCID SHO mice, each bearing a PSMA-positive PC3 tumor on one flank and aPC3 wild-type tumor on the other. For the PCa tumor xenografts, ˜10⁶cells were injected subcutaneously (sc) into each flank. Tumor formationoccurred after 2-4 weeks and was monitored using calipers andultrasound. A bio-distribution study was performed to obtain informationon the tumor uptake of the NPs. To this end, ca. 20 μCi of ⁸⁹Zr-DFOgrafted HBPE(CT20p) NPs conjugated to the lead PSMA targeting ligandswere injected into a cohort of mice and the tumors and organs wereharvested 6, 12, 24, 48, 72 and 96 h after injection. The time point forthe following in vivo imaging studies were based on the bio-distributiondata. For PET imaging (Focus 120, CTI/Siemens), standard uptake values(SUV) were determined for the PSMA(+) and (−) tumors. Dose findingstudies were performed to obtain the minimal required dose for imaging,which are at around 125 μCi. The tumors were harvested forimmunohistochemistry to detect PSMA and to confirm co-localization ofthe probe via autoradiography. Based upon the specific activity of theNPs, the amount of NPs within the tumors was estimated. To assess thespecific binding of the NPs to PSMA, the following controls were used,(1) HBPE-NP without targeting ligand, (2) PMPA, as a known inhibitor ofPSMA, was co-injected with the NPs and (3) excess (non-conjugated) smallmolecule ligands were co-injected with the NPs as blocking experiments.A set of control experiments included mice bearing FR(+)/PSMA(−) tumors(such as MDA-MB 231 tumors) to test the specificity of the NPs for PSMAover FR.

Completion of Experiment 2 resulted in optimized polyglutamated folateDFO-HBPE (CT20p) NPs in the ˜80 nm range that were internalized by PCacells via PSMA, with minimal uptake by the FR, causing PCa-specific celldeath. PK studies in mice showed protection of CT20p in NPs and extendedcirculation of particles to yield optimal peptide dosing and toxicityinformation. The results from PET imaging indicate that polyglutamatedfolate ⁸⁹Zr:DFO-HBPE (CT20p) NPs principally localize to PSMA-expressingPCa tumors with little to no off-site targeting to FR(+) or PSMA(−)tumors or tissues like the liver or spleen.

The proposed polyglutamated folate peptides were synthesized by standardsolid-state peptide chemistry and obtained commercially. The peptidesynthesis procedures to build both the γ-polyglutamated folate peptideas well as the standard α-linked version were commercially available aswere the conjugation procedures of β-alanine and folic acid. The use ofSPDP to link a cysteine-containing peptide to NPs has been performed. Inthe unlikely event that use of SPDP proves unsatisfactory, select“click” chemistry. In this application, polyglutamated folate peptideswith a C-terminus azide (N₃) modification are used to conjugate topropargylated functionalized HBPE-NPs. This conjugation chemistry hasbeen used. Recently developed click chemistry reactions that do not useCu for this conjugation are now commercially available (Sigma) and canbe used as alternatives (Baskin, J. M., et al., Copper-free clickchemistry for dynamic in vivo imaging. Proceedings of the NationalAcademy of Sciences of the United States of America 104, 16793-16797(2007); Soriano Del Amo, D., et al., Biocompatible copper(I) catalystsfor in vivo imaging of glycans. Journal of the American Chemical Society132, 16893-16899 (2010)). In the unlikely event that the glutamatedfolate-peptides fail to achieve selective PSMA-targeting, an alternativeapproach using glutamate ureas can be employed and the synthesis of thisapproach can be optimized (Chen, Y., et al., Synthesis and biologicalevaluation of low molecular weight fluorescent imaging agents for theprostate-specific membrane antigen. Bioconjugate chemistry 23, 2377-2385(2012)). Also where PK studies show limited circulation of peptides orNPs, modified PEG chains can be conjugated to the NPs.

The NP experiments disclosed herein result in data on optimized reactionconditions. Data was tabulated and statistical analysis was performed asdescribed in Experiment 1.

Experiment 3. Examination of the Anti-Cancer Activity of PolyglutamatedFolate-HBPE-DFO[CT20p]-NPs in Metastatic PCa

The potential value of the PSMA-targeted peptide/NP platform to regressPCa tumors and impair metastatic disease was investigated in mousemodels of PCa and bone metastasis.

The disclosed studies had significant health benefits for patients withCRPC by providing a therapeutic agent that impairs metastasis withoutthe side effects associated with current treatment approaches as well asproviding a noninvasive method for imaging treatment outcomes inpre-clinical studies.

PCa may grow slowly for many years; but in time the cancer invadesneighboring tissue and enters circulation to metastasize at distantsites. For advanced PCa, hormone therapy results in positive responsesrates of 80-90%. However, most men eventually develop progressivedisease or CRPC following hormone therapy and usually suffer severe sideeffects from treatments, which can include impotence, incontinence,heart disease and osteoporosis (Schrecengost, R. et al., Molecularpathogenesis and progression of prostate cancer. Seminars in oncology40, 244-258 (2013)). Detection of early metastasis remains one of thechallenges for PCa due to the highly variable time frame of metastasisoccurrence for the post-treatment patient. Current therapies for CRPCand patients with metastatic disease usually target hormone (androgen)synthesis or signaling (e.g. abiraterone, enzalutamide). Theseapproaches are not curative and only extend for a short period(Leibowitz-Amit, R. et al., Targeting the androgen receptor in themanagement of castration-resistant prostate cancer: rationale, progress,and future directions. Curr Oncol 19, S22-31 (2012); Leibowitz-Amit, R.et al., The changing landscape in metastatic castration-resistantprostate cancer. Current opinion in supportive and palliative care 7,243-248 (2013)). To address these problems, targeted therapeutics areneeded that allow for the specific delivery and concentration of drugsto tumors localized to the prostate as well as to metastatic sites, mostcommonly in the lymph nodes or bone, while causing minimal damage tohealthy (non-transformed) tissue. Hampering treatment outcomes is thefact that monitoring of drug delivery and assessment of therapeuticefficacy, using existing imaging technologies in the clinic, isdifficult due to the low metabolic rate of PCa and the close proximityof the prostate to the bladder. Such problems limit the use of standardPET imaging with ¹⁸F-FDG, since the tracer accumulates in the bladderimmediately above the prostate, thus obscuring its evaluation. Thedevelopment of a targeted molecular nanotheranostic (dual therapy anddiagnostic) platform that delivers and concentrates therapeutic agentsin PCa tumors, and integrates the capacity for imaging, provides a muchneeded therapeutic approach for patients with CRPC. A NP platform isideal as NPs are long-circulating agents that, when properly decoratedwith targeting ligands that bind to cancer cell receptors, displayminimal liver accumulation, renal excretion or localization of cargo,like imaging agents, to the bladder. Disclosed herein is a targeted NPplatform developed to encapsulate a therapeutic peptide (CT20p) andendow the NPs with PET imaging capabilities in order to monitorbio-distribution and efficacy in murine models of PCa and metastaticdisease.

The CT20 Peptide and Targeting to PSMA

CT20p is a small lipophilic peptide based on the α9 helix of Bax.Importantly, CT20p has properties that are distinct from the parentprotein. Using biophysical and cell biology methods, it was shown thatCT20p formed a type of pore in simple lipid membranes (Garg, P., et al.,Transmembrane pore formation by the carboxyl terminus of Bax protein.Biochimica et biophysica acta 1828, 732-742 (2013); Tatulian, S. A., etal., Molecular basis for membrane pore formation by Bax protein carboxylterminus. Biochemistry 51, 9406-9419 (2012)). Expression or introductionof CT20p in cancer cells resulted in mitochondrial localization of thepeptide followed by cell death that was different from the parentprotein in that Bcl-2 overexpression, Bax deficiency or caspaseinhibition minimally blocked it (Boohaker, R. J., et al. RationalDevelopment of a Cytotoxic Peptide To Trigger Cell Death. Molecularpharmaceutics (2012)). This indicated that CT20p did not trigger theconventional mitochondrial apoptotic pathway that is frequently mutatedin cancer cells. It is shown herein that CT20p preferentially targetsmitochondria within cancer cells, causing clustering of theseorganelles. This reduces energy production which is required for thecytoskeleton to mediate adhesion and motility, leading to celldetachment and death (anoikis). These effects were not observed innormal cells, such as fibroblasts, normal epithelia and macrophages,since the mitochondria of non-transformed cells are less susceptible tothe lethal effects of CT20p. This is highly significant as theadministration of traditional drugs to treat cancer causes debilitatingside effects due to their off-target toxicity (Tolaney, S. M., et al.,Lymphopenia associated with adjuvant anthracycline/taxane regimens.Clinical breast cancer 8, 352-356 (2008)). Unlike these traditionaldrugs, the cancer-selective activities of CT20p block invasiveness andprevent metastasis without damaging normal cells.

Encapsulation of CT20p into HBPE-NPs facilitated the delivery of thepeptide to PCa cells. HBPE-NPs were perfectly suited for this task asthese can encapsulate multiple cargos within their hydrophobicnanocavities, without affecting the distribution of targeting ligands onthe NPs' surface. An encapsulation efficacy of CT20p within HBPE-NPs of95% was achieved, with particle stability at physiological pH andrelease of the peptide at pH<5. To deliver CT20p to PCa cells,especially metastatic cells, the peptide was encapsulated withinHBPE-NPs that were functionalized with polyglutamated folates.Polyglutamated folates are an innovative way to target PSMA, aPCa-specific cell surface protein highly expressed in PCa tumors but notnormal prostate (Bostwick, D. G., et al., Prostate specific membraneantigen expression in prostatic intraepithelial neoplasia andadenocarcinoma: a study of 184 cases. Cancer 82, 2256-2261 (1998);Israeli, R. S., et al., Molecular cloning of a complementary DNAencoding a prostate-specific membrane antigen. Cancer research 53,227-230 (1993); Ross, J. S., et al. Correlation of primary tumorprostate-specific membrane antigen expression with disease recurrence inprostate cancer. Clinical cancer research: an official journal of theAmerican Association for Cancer Research 9, 6357-6362 (2003); Silver, D.A., et al., Prostate-specific membrane antigen expression in normal andmalignant human tissues. Clinical cancer research: an official journalof the American Association for Cancer Research 3, 81-85 (1997)). PSMAexpression correlates with androgen independence and increasedmalignancy of PCa (Wright, G. L., Jr. et al. Upregulation ofprostate-specific membrane antigen after androgen-deprivation therapy.Urology 48, 326-334 (1996)). Most importantly PSMA is overexpressed inPCa metastatic lesions, facilitating the targeting of the therapeutic NPto metastatic sites like bone or lymph nodes (Chang, S. S., et al. Fivedifferent anti-prostate-specific membrane antigen (PSMA) antibodiesconfirm PSMA expression in tumor-associated neovasculature. Cancerresearch 59, 3192-3198 (1999); Milowsky, M. I., et al. Vascular targetedtherapy with anti-prostate-specific membrane antigen monoclonal antibodyJ591 in advanced solid tumors. J Clin Oncol 25, 540-547 (2007); Morris,M. J., et al. Phase I evaluation of J591 as a vascular targeting agentin progressive solid tumors. Clinical cancer research: an officialjournal of the American Association for Cancer Research 13, 27072713(2007)). Polyglutamated folates specifically target PSMA on PCa cellsand not the folate receptor (FR) (found on cells like macrophages)because PSMA exhibits glutamate carboxylase as well as folate hydrolaseactivities, hydrolyzing extracellular polyglutamated folate tomono-glutamic folic acid that is utilized by cells (Ghosh, A., et al.,Tumor target prostate specific membrane antigen (PSMA) and itsregulation in prostate cancer. J Cell Biochem 91, 528-539 (2004)).Upregulation of PSMA provides PCa cells with a growth advantage in thelow folate tumor environment, preventing downregulation of PSMA andensuring a stable target for the HBPE-NPs encapsulating CT20p (Yao, V.,et al., Prostate specific membrane antigen (PSMA) expression givesprostate cancer cells a growth advantage in a physiologically relevantfolate environment in vitro. The Prostate 66, 867-875 (2006); Yao, V.,et al., Expression of prostate-specific membrane antigen (PSMA),increases cell folate uptake and proliferation and suggests a novel rolefor PSMA in the uptake of the non-polyglutamated folate, folic acid. TheProstate 70, 305-316 (2010)). The disclosed compositions and methodstake advantage of PSMA's binding affinity towards polyglutamated folateto develop a NP platform technology for delivery of the CT20p to PCa.

A NP platform was developed that targets PCa cells via PSMA to deliver atherapeutic peptide and incorporates imaging capabilities to facilitatepre-clinical bio-distribution, pharmacokinetics (PK) and efficacystudies. The major innovative feature is the combination of an optimizedNP design that protects and delivers a therapeutic cargo, CT20p, tocancer cells, and a targeting scheme that relies on a biomarker, PSMA,which is unique to high grade and metastatic PCa. In pre-clinicalmodels, similar ligand-targeted NPs provided benefits in terms of targetcell internalization and retention (van der Meel, R., et al.,Ligand-targeted particulate nanomedicines undergoing clinicalevaluation: current status. Advanced drug delivery reviews 65, 1284-1298(2013)). However, the ligand-targeted HBPE-NPs disclosed herein are animprovement over other nanomaterials because these do not produce toxiceffects, such as the generation of reactive oxygen species (ROS),induction of autophagy or lysosomal degradation, associated withparticles made from iron oxide, silica or titanium (Stern, S. T., etal., Autophagy and lysosomal dysfunction as emerging mechanisms ofnanomaterial toxicity. Particle and fibre toxicology 9, 20 (2012). Theplatform disclosed herein is distinct from nanomedicines, such asliposomes, in terms of the composition of the HBPE-NPs and, mostimportantly, its cargo (CT20p), which unlike other drug payloads (e.g.Doxorubicin or Taxol) is cancer-cell specific with little to nocytotoxicity to non-cancerous tissues as is described herein.

Polymeric NP Design for Dual Therapy and Imaging

In one aspect, the surface of the NPs disclosed herein comprisedcarboxylic acid groups that enable functionalization with targetingligands. These NPs were fabricated from an aliphatic HBPE polymer whichwas modified to graft chelating ligands (e.g., DTPA, DOTA or DFO) forPET imaging capabilities (Santra, S., et al., Aliphatic hyperbranchedpolyester: a new building block in the construction of multifunctionalnanoparticles and nanocomposites. Langmuir 26, 5364-5373 (2009)). Thedesigned HBPE polymers have major advantages over conventional linearpolymers (such as PLGA) since: (i) they are highly branched, creatingunique hydrophobic cavities; (ii) they display numerous surfacecarboxylic acid groups for facile labeling of targeting ligands, and(iii) monomers contain an acidic proton that can be easily displaced bya pendant ligand to achieve further functionalization of the NP'scavities, such as introducing a chelating ligand for stableencapsulation of radioactive isotopes for PET imaging. Note that currentlinear polymers fail to generate well-defined hydrophobic nanocavitiesand are, thereby, difficult to modify chemically and introduce multiplefunctionalities. For example, dendrimers, although highly branched, formnanocavities that are difficult to synthesize and chemically engineer tointroduce imaging functionalities. Hence, a strong innovative aspect ofthe approach disclosed herein is the use of the HBPE polymer tofabricate a multifunctional theranostic polymeric NP targeting a PCaspecific protein, PSMA, while chemically engineering the particle'snanocavities to incorporate chelating agents for PET imaging. While thePET imaging feature of the HBPE-NPs is for the assessment of thenanoagent's bio-distribution in mice, the present disclosure advancesthe translational use of PET to assess the delivery of therapeutics inpatients. To endow the NPs with PET imaging capabilities,Desferrioxamine (DFO) was grafted onto the HBPE nanocavities. DFO is achelating agent that strongly binds Zr and is used in the design of⁸⁹Zr-PET imaging probes (Kiss, T. et al., Metal-binding ability ofdesferrioxamine B. Journal of Inclusion Phenomena and MolecularRecognition in Chemistry 32, 385-403 (1998)). By introducing a pendantgroup into the hydrophobic cavities with selective binding to ⁸⁹Zr, theability of the HBPE-NPs to chelate ⁸⁹Zr and also encapsulate thetherapeutic peptide, CT20p, while displaying targeting ligands wasincreased; thus creating a NP platform to assess the delivery of atherapeutic peptide by PET imaging. The use of ⁸⁹Zr as a PET tracer isgaining acceptance as a long-lived positron emitter radioisotope for thedetection of tumors by PET (Meijs, W. E., et al. Zirconium-labeledmonoclonal antibodies and their distribution in tumor-bearing nude mice.Journal of nuclear medicine: official publication, Society of NuclearMedicine 38, 112-118 (1997); Verel, I., et al., 89Zr immuno-PET:comprehensive procedures for the production of ⁸⁹Zr-labeled monoclonalantibodies. Journal of nuclear medicine: official publication, Societyof Nuclear Medicine 44, 1271-1281 (2003)). The ⁸⁹Zr radionuclide hasmultiple advantages over ⁶⁴Cu radionuclide such as: (1) a half-life ofapproximately 78.4 h (3.17 days) as opposed to the 12.7 h for the ⁶⁴Cuisotope, (2) a positron yield of 22.7% which improves countingstatistics when compared to other radioisotopes, (3) no known toxicityto biological systems, and (4) generation of ⁸⁹Zr is cost effective andhighly efficient. Recently, the use of ⁸⁹Zr-labeled antibodies to imageHER2/neu-positive (Holland, J. P., et al., Measuring the pharmacodynamiceffects of a novel Hsp90 inhibitor on HER2/neu expression in mice usingZr-DFO-trastuzumab. PLoS One 5, e8859 (2010)) and PSMA-positive(Ruggiero, A., et al., Targeting the Internal Epitope ofProstate-Specific Membrane Antigen with ⁸⁹Zr-7E11 Immuno-PET. Journal ofnuclear medicine: official publication, Society of Nuclear Medicine 52,1608-1615 (2011)) tumors in vivo was reported and the potential clinicaluse of this radiotracer for localizing these tumors was suggested. As⁸⁹Zr has a long half-life (3.17 days), it is appropriate forencapsulation into long circulating NPs.

In another aspect, disclosed are the design and screening ofpolyglutamated folate peptides as targeting ligands to PSMA. PSMA is avalidated target to deliver imaging and therapeutic agents to PCa. Ananti-PSMA monoclonal antibody (mAb) was used to image and deliverchemotherapeutics directly to PCa; however this approach, while proof ofprincipal, was suboptimal with low sensitivity to detect viable tumors(Freeman, L. M., et al. The role of (111) In Capromab Pendetide(Prosta-ScintR) immunoscintigraphy in the management of prostate cancer.Q J Nucl Med 46, 131-137 (2002); Haseman, M. K., et al., CapromabPendetide imaging of prostate cancer. Cancer Biother Radiopharm 15,131-140 (2000); Horoszewicz, J. S., et al., Monoclonal antibodies to anew antigenic marker in epithelial prostatic cells and serum ofprostatic cancer patients. Anticancer Res 7, 927-935 (1987); Lopes, A.O., et al., Immunohistochemical and pharmacokinetic characterization ofthe site-specific immunoconjugate CYT-356 derived from antiprostatemonoclonal antibody 7E11-C5. Cancer research 50, 6423-6429 (1990);McDevitt, M. R., et al., An alpha-particle emitting antibody([213Bi]J591) for radioimmunotherapy of prostate cancer. Cancer research60, 6095-6100 (2000); Smith-Jones, P. M., et al., Radiolabeledmonoclonal antibodies specific to the extracellular domain ofprostate-specific membrane antigen: preclinical studies in nude micebearing LNCaP human prostate tumor. Journal of nuclear medicine:official publication, Society of Nuclear Medicine 44, 610-617 (2003)).As alternative to antibodies, PSMA-binding aptamers were identified andconjugated to polymeric NPs, encapsulating the anticancer drug,Docetaxel, for the targeted treatment of LNCaP xenografts in nude mice(Cheng, J., et al., Formulation of functionalized PLGA-PEG nanoparticlesfor in vivo targeted drug delivery. Biomaterials 28, 869-876 (2007);Farokhzad, O. C., et al., Targeted nanoparticle-aptamer bioconjugatesfor cancer chemotherapy in vivo. Proceedings of the National Academy ofSciences of the United States of America 103, 6315-6320 (2006);Farokhzad, O. C., et al., Nanoparticle-aptamer bioconjugates: a newapproach for targeting prostate cancer cells. Cancer research 64,7668-7672 (2004)). However, these studies were not reproducible due tostability issues with the aptamers in serum. Moreover, while some NPformulations, using antibodies and aptamers to target PSMA, arecurrently in Phase I clinical trials, these NPs do not possess imagingcapabilities (Hrkach, J., et al., Preclinical development and clinicaltranslation of a PSMA-targeted docetaxel nanoparticle with adifferentiated pharmacological profile. Science translational medicine4, 128ra139 (2012)). In addition, other targeting ligands for PSMAinclude glutamated ureas; which further validates the use of glutamateconjugates of folate to target PSMA (Barrett, J. A., et al.,First-in-man evaluation of 2 high-affinity PSMA-avid small molecules forimaging prostate cancer. Journal of nuclear medicine: officialpublication, Society of Nuclear Medicine 54, 380387 (2013); Chen, Y., etal., Radiohalogenated prostate-specific membrane antigen (PSMA)-basedureas as imaging agents for prostate cancer. J Med Chem 51, 7933-7943(2008)). However, the complex synthesis of glutamated ureas hamperstheir widespread use and study. Hence, while targeting PSMA had beenvalidated in other studies, the use of effective ligands for PSMAremained to be developed and the incorporation of imaging agents had notbe achieved. To address this problem, a method to target NPs to PSMA isdisclosed herein. Considering that PSMA utilizes polyglutamated folateas its biological ligand and it is shown herein that folicacid-conjugated HBPE-NPs target PSMA, a series of polyglutamated folatepeptides were conjugated on the HBPE-NP surface for targeting PSMA.Screening of these polyglutamated folate peptides-NP formulationsidentified a conjugate with higher and more specific binding toward PSMAthan folate alone with minimal binding to the folate receptor (FR). Theapproach disclosed herein is significantly different from others thattargeted PSMA since polyglutamated folate peptides were used to directNPs to PSMA-expressing PCa cells. As these peptides are more stable andeasier to manufacture than monoclonal anti-PSMA antibodies, members ofthe resulting multivalent HBPE-NP library provide a more robustPSMA-targeting nanoplatform to target PCa. Finally, as PSMA is not onlyexpressed in the primary tumor, but also in metastatic lesions, itfacilitated the delivery of CT20p to treat the primary tumor and alsoany metastasis.

Cancer-Specific Therapeutic Peptide with Anti-Metastatic Activity

In another aspect, disclosed is the therapeutic peptide, CT20p(Boohaker, R. J., et al., The use of therapeutic peptides to target andto kill cancer cells. Current medicinal chemistry 19, 3794-3804 (2012)).This peptide induces cancer-specific cell death. Currently biologicalslike CT20p account for ˜30% of drugs being tested and about half of newmolecular entities. However, the mechanism of action for manybiologicals, such as anti-microbial peptides, remains mostly unknown,challenging the identification of target patient populations. CT20p isan improvement in that it works in the nanomolar range, and the methodby which it exerts its biological activity on cells is disclosed herein.The cancer-specificity of CT20p is based on its effects uponmitochondrial dynamics and the cytoskeleton. This powerfulcancer-directed action of CT20p impairs the invasiveness that underliesthe transition to metastasis, indicating that the peptide is ideal foruse in cancer patients with disseminated disease, such as can resultfrom CRPC. In addition, the ability of CT20p to spare normal cells lieswith the fact that the peptide takes advantage of documented differencesin mitochondrial physiology unique to cancer cells (Desai, S. P., etal., Mitochondrial localization and the persistent migration ofepithelial cancer cells. Biophysical journal 104, 2077-2088 (2013);Zhao, J., et al., Mitochondrial dynamics regulates migration andinvasion of breast cancer cells. Oncogene 32, 4814-4824 (2013)).Disclosed herein is the first study to develop a therapeutic peptidewith anti-metastatic properties that can be encapsulated in HBPE-NPs,specifically targeted to PCa via PSMA. As shown in FIG. 1, theuniqueness of the HBPE-NP-CT20p platform extends to its ability toefficiently escape endosomes. Ligand-targeted HBPE-NPs were internalizedthrough receptor-mediated endocytosis. The HBPE-NPs protected thepeptide through the endocytic pathway, destabilizing at the acidic pH oflate endosomes-lysosomes as shown herein. CT20p (which contains chargedresidues) then escaped from the endosomes, potentially by forming a porein the endosome membrane was showed with lipid vesicles, and thenchaperones, like HSP90, facilitated translocation to the cytosol as wasreported with other endosome-localized proteins (e.g. toxins (Rafts, R.,et al., The cytosolic entry of diphtheria toxin catalytic domainrequires a host cell cytosolic translocation factor complex. The Journalof cell biology 160, 1139-1150 (2003)) or growth factors (Wesche, J., etal., FGF-1 and FGF-2 require the cytosolic chaperone Hsp90 fortranslocation into the cytosol and the cell nucleus. The Journal ofbiological chemistry 281, 11405-11412 (2006)). Once in the cytosol,CT20p then associated with mitochondria.

Engineering of Polymeric NP

To endow HPBE-NPs with imaging capabilities, the synthesis procedure wasmodified to generate an epoxy-grafted polymer that producedFe(III)-DFO-grafted or Gd(III)-DTPA-grafted HBPE polymers (FIG. 16).Briefly, diethylmalonate (1) (62.5 mmol), 3-chloroprop-1-ene (62.5 mmol)and potassium carbonate (312.5 mmol) were taken in acetonitrile andrefluxed. Stoichiometric amounts of chloroprop-1-ene and potassiumcarbonate, as a mild base, facilitated the release of one acidic protonfrom 1. The resulting monoalkylated product (40.0 mmol), was purified byflash chromatography and reacted with 4-bromobutyl acetate (48 mmol) ina dry THF solution containing NaH (56 mmol). In this second step, theuse of NaH as a stronger base and the excess amount of 4-bromobutylacetate ensured the removal of the second acidic proton and theformation of the dialkylated compound 2. Deprotection of 2 (19.2 mmol),by hydrolysis of the protecting ester groups in an aqueous methanolsolution containing NaOH (67.3 mmol), resulted in the formation ofmonomer 3, containing a propene group as a pendant ligand. Monomer 3 waspolymerized using p-toluenesulfonic acid (100:1 molar ratio) ascatalyst. The resulting propene-grafted polymer 4 was oxidized to anepoxide to make it reactive to the terminal amine group in DFO orDTPA-amine. Briefly, 3-chloroperoxybenzoic acid (1.2 mmol) was dissolvedinto a mixture of dry dichloromethane (DCM) and Na₂CO₃ (1.2 mmol). Tothis, the polymer 4 (120 mg) dissolved in dry DCM was added. Theoxidized polymer was precipitated in water to obtain the epoxy-graftedpolymer intermediate 5. This reactive epoxy-grafted polymer 5 generatedeither an Fe(III)-DFO- or the Gd(III)-DTPA-grafted HBPE by reacting theepoxy polymer (40 mg) with the corresponding chelator (0.122 mmol) in amethanol solution containing triethylamine (0.203 mmol). GPC analysisindicated an average polymer molecular weight of 40 kDa. To assess theNP bio-distribution by PET, the DFO-grafted HBPE-NPs that encapsulate⁸⁹Zr were studied. This route was chosen as PET is more sensitive thanMRI and is the imaging modality typically used in biodistributionstudies. In FIG. 3, the DFO-grafted HBPE-NPs were fabricated using aFe³⁺ chelated DFO to facilitate “wrapping” of the DFO around the metalfor a better fit in the NP's inner cavities via the solvent diffusionmethod (FIG. 17A). Under these conditions, the miscible solvent rapidlydiffused into the water, causing the polymer to self-assemble, formingpolymeric NPs encapsulating molecules within hydrophobic pockets. Thisprocess exposed the hydrophilic segments of the polymer to the aqueoussolution, resulting in the formation of carboxyl-functionalized NPsencapsulating a near-infrared dye, chelated metal and CT20p.

HBPE-NPs surface functionalization with folate ligands was performedusing standard conjugation procedures (Kaittanis, C., et al., Role ofnanoparticle valency in the nondestructive magnetic-relaxation-mediateddetection and magnetic isolation of cells in complex media. Journal ofthe American Chemical Society 131, 12780-12791 (2009); Santra, S., etal., Drug/dye-loaded, multifunctional iron oxide nanoparticles forcombined targeted cancer therapy and dual optical/magnetic resonanceimaging. Small 5, 1862-1868 (2009); Santra, S., et al., Cell-specific,activatable, and theranostic prodrug for dual-targeted cancer imagingand therapy. Journal of the American Chemical Society 133, 16680-16688(2011); Santra, S. et al., Selective N-Alkylation of beta-AlanineFacilitates the Synthesis of a Poly(amino acid)-Based TheranosticNanoagent. Biomacromolecules (2011)). To conjugate folic acid to theHBPE-DFO(CT20p)-NPs, folic acid was reacted with ethylene diamine toyield aminated folate (Folate-NH2), which was reacted to the carboxylicacid groups on HBPE-DFO(CT20p)-NPs via carbodiimide chemistry, formingthe folate conjugated HBPE-DFO(CT20p)-NPs. Scanning transmissionelectron microscopy (STEM) showed that these NPs were, on average,monodispersed NPs of 80 nm in diameter (FIG. 17B). As CT20p is ahydrophobic peptide, it can be encapsulated within the hydrophobicpockets of the hyperbranched polymer during NP formation with anencapsulation efficacy of 95%. The CT20p peptide cargo within the NP wasstable at physiological pH (˜pH 7) and only released from the NP at pH(<pH5) (FIG. 17C). Upon incubation with cold Zr⁴⁺ (in the form ofZrCI₄), the chelated Fe³⁺ was easily displaced by Zr⁴⁺. This wascorroborated by ICP-MS results, showing a percent by weight of Zr⁴⁺ topolymer of 0.15% in the final HBPE-DFO(CT20p)-NP formulation. No Zr wasdetected in control HBPE-NPs, which indicated that the NPs without DFOdid not chelate Zr non-specifically. Mass spectrometry studies of DFOand DFO:Fe, incubated with Zr, showed that both can chelate Zr (FIG.14A) and that the DFO:Fe can exchange the Fe for Zr. Visual confirmationof Fe-chelation and displacement by Zr³⁺ was observed as an intenseorange coloration in the Folate-HBPE-DFO(CT20p)-NPs that occurred uponFe addition and disappeared upon subsequent addition of Zr (FIG. 14B).The presence of Zr in the polymer was corroborated by ICP-MS. Theseresults provide a reliable way to label the Folate-HBPE-DFO(CT20p)-NPswith ⁸⁹Zr for PET imaging right before the animal studies. Therefore,the HBPE-NPs can be tailored to chelate a radioisotope for PET imagingthat, when delivered to PCa cells, produce a targeted PET imaging agentfor assessment of drug bio-distribution.

Peptide Cargo with Cancer Specific Mitotoxic and Anti-Adhesion Activity

Recently, a mitotoxic peptide that kills cancer cells was discovered. Tovisualize the cytosolic localization of CT20p (delivered to cells byHBPE-NPs), rhodamine-labeled CT20p was used, which proved equallyeffective in killing as compared to the unlabeled peptide. Usingnanomolar amounts of CT20p (˜3.4 nM) in NPs, co-localization of thepeptide (red) with mitochondria (green) was detected in cancer cells(yellow fluorescent overlay) (FIG. 18A), and no other organelles such asthe ER. This triggered hyperpolarization of the mitochondrial membraneand fusion-like aggregation (FIG. 18B) that impaired mitochondrialredistribution, ATP production and, as a result, F-actin polymerization(FIG. 18C). These “initiating events”, during which cells remainedviable (FIG. 18D), resulted in cell detachment, starting at 6 hourspost-treatment (FIG. 18E), which was preceded by decreased surfaceexpression of β1 integrin, the adhesion molecule that (along with α5)mediates binding to the fibronectin substrate (FIG. 18F). By 24 hourspost-CT20p treatment, “effector events” caused by peptide-induced lossof substrate attachment were detected, including the activation ofcaspases (FIG. 18G), the induction of autophagy (FIG. 18H), andincreased ROS production (FIG. 18I). Note that none of these “effector”events were detected earlier or upon treatment with HBPE-NPs alone,indicating that CT20p, and not the NPs, caused these effects. Cell death(anoikis), indicated by membrane asymmetry, was detectable in cancercells by 48 hours (FIG. 18J). Similar results, such as mitochondriallocalization, autophagosome formation or cell death, were not observedwith a control epithelial cell line (FIGS. 19A-19C), indicating that theHBPE-NPs did not cause non-specific effects and that the lethal activityof the peptide was linked to cancer cells. These findings indicate thatCT20p impairs cancer cell invasiveness through its actions onmitochondria and the cytoskeleton, which causes detachment-induced celldeath.

Folate-HBPE-NPs Target PSMA in PCa Cells

To visualize NP internalization into PSMA-expressing PCa cells, afluorescent dye (DiI) was encapsulated within folate-HBPE-NPs.Cell-associated fluorescence was detected by flow cytometry andfluorescence microscopy in LNCaP (PSMA+) and PSMA(+)-PC-3 cells (FIG.20). No internalization was observed in PSMA(−) PC-3 cells or in a PSMAinhibitor (PMPA) (FIG. 20). These results indicated thatfolate-conjugated NPs target PSMA and release a therapeutic cargo insidethe cell. Next, folate-HBPE(CT20p)-NPs were synthesized to deliver CT20pto PCa cells via PSMA. Upon incubation of LNCaP cells withfolate-HBPE(CT20p)-NP, a dose and time dependent response was observed,achieving cell death after 48 hours with nanomolar amounts of peptide inNPs (FIGS. 21A, 21D). Similar results were obtained with PSMA(+) PC-3cells (FIGS. 21B, 21E). No cytotoxicity was observed in PSMA(−) PC-3cells (FIGS. 21C, 21F) or when PMPA was used as PSMA inhibitor. Theseresults show that the folate-decorated HBPE-NPs deliver CT20p to PCa viaPSMA, achieving target specific cell death. Fluorescence microscopy ofLNCaP cells using folate-HBPE(CT20p/DiI)-NP, showed significant cellassociated DiI fluorescence, causing initial detachment and then deathwithin 48 hrs (FIG. 21G), as measured by the uptake of a membranepermeability dye (Sytox) (FIG. 21H). Note that studies using controls,such as free CT20p (not taken up by cells) and untargeted HBPE-NPs todeliver CT20p (less effective), are known in the art.

To study any effect that folate-HBPE (CT20p)-NPs may have onnon-transformed cells, macrophages (RAW cells) were incubated with thefolate-HBPE(CT20p)-NPs and cell death was assessed. FIGS. 21I and 21Jshow that macrophages were minimally affected when incubated withfolate-HBPE(CT20p)-NPs for 48 hours (<4% dead) when compared tountreated cells (FIGS. 21I and 21J, left panels). As a control, thedeath of macrophages was observed upon treatment with the Folate-ss-Doxoprobe or Doxorubicin (FIGS. 21I and 21J, right and middle panels). Theseresults showed that Folate-HBPE (CT20p)-NPs are not toxic tomacrophages, while inducing cell death in cancer cells. Fluorescencemicroscopy studies confirmed the internalization offolate-HBPE(CT20p)-NPs by macrophages, indicating that whenCT20p-containing NPs are taken up little change in the viability ofnontransformed cells is observed.

Next, PEGylated folate-HBPE-NPs were synthesized to examine in vivoPSMA-specific targeting using mice bearing right flank PSMA(+) PC3 andleft flank PSMA(−) PC3 tumors. Tumors were allowed to grow for 2 weeksbefore treatment with one intravenous (IV) injection offolate-HBPE(DiR)-NPs, containing a near infrared dye (DiR) (2mg/kg/dose). After 24 hrs, mouse fluorescence imaging showed a strongfluorescence signal in the PSMA(+) PCa tumors (FIG. 22A), while nofluorescence was observed in wild type PC3 tumors that lack PSMA. Thisexperiment was repeated twice to confirm that the fluorescent signal wasrestricted to the PSMA+ tumors. These results demonstrated thatfolate-conjugated HBPE-NPs target PSMA expressing PCa tumors, withminimal off-target accumulation and that PEGylation of NPs does notinterfere with ligand targeting. Next, the anti-tumor effect of thePSMA-targeting, PEGylated folate-HBPE(CT20p)-NPs was evaluated in micebearing PSMA(+) and PSMA(−) PC3 tumors. A single IV treatment withCT20p-containing NPs (2 mg/kg/dose or ˜3.4 nM CT20p) caused significantregression of the PSMA-targeted tumors (FIG. 22B). Note that after 10days, growth of PSMA-targeted tumors did not recur. Histologicalexamination of tissues by a pathologist revealed fragmentation and areasof necrosis in the PSMA+ tumors not evident in the untargeted tumors orin the liver and spleen, (FIG. 22C). In the graph shown in (FIG. 22D), asummary of a two week mouse experiment shows that PEGylatedFOL-HBPE(CT20p)-NPs, IV, injected once a week (2 mg/kg/dose or ˜3.4 nMCT20p), effectively concentrated CT20p in PSMA+ tumors. UntargetedCOOH-NPs also delivered CT20p to tumors, likely through the enhancedpermeability and retention (EPR) effect, but efficacy was less due toreduced amounts in tumors and accumulation in the liver and spleen(detected by DiR fluorescence,). Once delivered to tumors, CT20p is moreeffective than drugs like Doxoyrubucin (Dox) (FIG. 22D). In total, thedata indicates that PEGylated folate-HBPE-NPs deliver CT20p to PSMA(+)PCa cells and that particles persist in tumors, causing targeted tumorregression. This work provides the foundational support for the testingof the polyglutamate folate-HBPE-NPs as an improvement over thefolate-targeted NPs to concentrate CT20p in PSMA(+) PCa tumors andmetastatic sites.

In this experiment, the ability of polyglutamated folate-DFO-HBPE-NPs todeliver CT20p to PCa tumors and impair metastasis was investigated. Inaddition, the biological effect of CT20p upon the cytoskeleton andintegrin signaling that promotes its anti-metastatic activities as wellas the effectiveness of CT20p under hormone or castration conditionswere examined. The murine model systems used will include xenografts ofhuman PCa cell lines that express PSMA as well as PSMA-negative PCa celllines. The polyglutamated folate DFO-HBPE(CT20p)-NPs from Experiment 2that demonstrated the most selective PSMA targeting in PET imagingstudies was used. As a model of PCa metastasis, an orthotopic PCa modelin which luciferase expressing LNCap cell (LNCap-luc-M6) are implanteddirectly in the prostate was used (Scatena, C. D., et al., Imaging ofbioluminescent LNCaP-luc-M6 tumors: a new animal model for the study ofmetastatic human prostate cancer. The Prostate 59, 292-303 (2004)). Thismodel is ideal to study the progression of metastatic prostate cancer tolymph nodes and lungs by bioluminescence. In addition, as PCa primarilymetastasize to the bones, a method where PCa bone (osseous) tumors areestablished by injecting PCa cells directly into a mouse tibia was used(Ulmert, D., et al., Imaging androgen receptor signaling with aradiotracer targeting free prostate-specific antigen. Cancer discovery2, 320-327 (2012)). In addition, results using Doxorubicin (Doxo) orFolate-s-s-Doxo were compared to those obtained with polyglutamatedfolate HBPE(CT20p)NPs. The results indicated that polyglutamated folateHBPE-DFO[CT20p]-NPs target PSMA-expressing PCa cells, causing loss ofcell adhesion and invasiveness, which will impair the development ofmetastasis.

Examining the Effects of CT20p Upon Cell Adhesion and Anoikis

An essential activity that promotes the invasiveness of metastatic cellsis rearrangement of the cytoskeleton, which can be linked to integrinsignaling. To demonstrate that CT20p alters this activity in PCa cells,the distribution of F-actin and G-actin with mitochondria (whichproduces the ATP that powers actin polymerization) in cells treated withpolyglutamated folate HBPE-DFO[CT20p]-NPs (lead compounds fromExperiment 2) was examined. At selected time points, PCa cells (Table 1)as well as control cells were treated with Mitotracker Red to visualizemitochondria and then were fixed on coverslips for staining with AlexaFluor™ 647 phalloidin, a high-affinity F-actin probe, orDeoxyribonuclease I, Alexa Fluor™ 488-conjugate for G-actin for imagingby confocal microscopy (Zeiss 710). To examine how integrin signalingwas impaired by CT20p, the surface expression of integrins detected withfluorescence-conjugated antibodies (β1, CD29 and αVβ3, CD51/61) by flowcytometry, was examined. Levels of additional integrins (αV or α5) werealso evaluated by immunoblotting cell lysates. To determine whether celldetachment and the reduction in integrin levels could activate anoikis,the CytoSelect® 96-well Anoikis Assay Kit was used and standard assaysof cell migration and invasion were performed. Additionally, to test theeffects of CT20p under conditions of directed migration, the cellsdescribed above were treated with conditioned media from 3T3 cells,which contains chemotactic factors.

Assessment of Tumor Regression Under Hormone and Castration Conditions

Since a hallmark of CRPC is the re-activation of androgen signaling,even after castration, it is important to test the effectiveness ofCT20p as well as the ability of the PSMA targeting DFO-HBPE-NPs todeploy a therapeutic payload (CT20p) directly to the PCa tumors undercastration or hormone signaling conditions. Results were compared withthose obtained with free doxorubicin or folate-ss-doxorubicin. Again,the lead HBPE-NP preparations from Experiment 2 were used. Male SCIDmice were used that are either intact or castrated and treated withtestosterone (SC) or DHT pellet. SCID mice were injected SC withluciferase expressing LNCaP cells and, upon tumor growth, D-luciferinwas injected prior to imaging (IVIS, Perkin Elmer). In all experiments,NPs without targeting ligand or without CT20p were used as controls.Mice were injected once every week, for 3-4 weeks, with controlDFO-HBPE-NPs or PSMA-targeted DFO-HBPE(CT20p)-NPs. Tumor growth wasmonitored by measuring the tumor size using a caliper or by ultrasoundas well as by bio-luminescence as described above. In addition, bloodwas collected for weekly PSA measurements (with a commercially availableELISA), using the value prior to therapy as a baseline. The tumors werefollowed for 6 weeks or until reaching 1.5 cm in size (whichever camefirst). At experimental endpoints, remaining tumor tissue, as well asliver, spleen, kidney, lungs and brain, were collected and examined byimmunohistochemistry (using J591 to identify PSMA) and also qRT-PCR andquantitative Western Blot for PSMA levels in the tumor to correlateswith response to the targeted therapy. Tissues were then mounted forhistological examination using H & E staining for detecting the presenceof malignancy, treatment effect (absence or presence of necrosis).

Targeting PCa Metastasis in Mice

A previously reported orthotopic PCa mouse model was used in theseexperiments.⁶⁰ This model was developed by injecting LNCap-luc-M6 cellsinto the dorsolateral prostate lobes of male SCID-bg-mice. This modelwas used in order to visualize metastatic cells by bioluminescence.Using this system, within a period of 16 weeks, a luciferase expressingPCa tumor developed in the prostate, as well as luciferase expressingmetastatic lesions in nearby lymph nodes and lungs. Approximately 10⁶LNCap-luc-M6 cells were injected directly into the prostate ofSCID-bg-mice and the mice were monitored weekly by bioluminescence afterinjection of D-luciferin to detect the development of primary tumors andmetastasis. Another set of mice were injected with PC3-luc2 as negativecontrol as these cell lines did not express PSMA. After development ofmetastatic PCa in these mice, PSMA targeting DFO-HBPE(CT20p)NPs wereinjected and survival data was acquired. In the event that the signalfrom the primary tumor is too bright and prevents detecting themetastatic cells, the primary tumor can be blocked or removed. Next,since PCa metastasis occur more commonly in the bone, the capabilitiesof the PSMA targeting DFO-HBPE(CT20p) NPs to target tumors seeded to thebone were examined. To create bone tumors, the tibiae of mice wereexposed and a small hole was drilled through the cortex into the marrowspace using a stero-microscope. Once the cavity was accessed,concentrated PSMA(+) PC3 cells were slowly injected. In this model, thePSMA(+) PC3 cells were used, as PC3 is a cell line derived from humanbone metastasis. After removal of back-flushed cells, the drill hole wasclosed with bone wax and the skin was closed with sutures. Once tumorswere detected by X-ray CT, the mice were injected with the correspondingNPs and imaged as described herein. The mice were sacrificed and thenumber and mean-size of metastases was correlated with the read outobtained by imaging. Controls comprised mice bearing PSMA(+) tumors butinjected with non-targeted DFO-HBPE-NPs. In addition, in another set ofmice the tibiae were injected with wild-type PC3 cells to developPSMA(−) bone metastasis as a negative control. Toxicity to non-canceroustissue was examined by histology and serum was recovered for clinicalchemistry tests of liver and kidney function as described in Experiment2.

Comparison with Free Doxo and Folate-s-s-Doxo and Folate HBPE (Doxo)

Some of the mouse tumor models described herein were injected with freeDoxo, Folate-s-s-Doxo or Folate HBPE (Doxo) (see FIGS. 22A-22D). Tumorregression also occurs with these therapeutics, but their correspondingefficacy in targeting the metastasis as well as any off-targeted tissuetoxicity was assessed and compared to results obtained with thepolyglutamated folate HBPE(CT20p) NPs. Toxicity to non-tumor tissue wasexamined as described herein.

Assessment of NP Localization by PET

To monitor localization of polyglutamated folate HBPE(CT20p) NPs to bothprimary and metastatic tumors, the theranostic version of the PSMAtargeted HBPE (CT20p)NPs (with ⁸⁹Zr) from Experiment 2 was used in someof the animal models described herein. PSMA expression levels atmetastatic sites and tumor regression were imaged by X-ray CT and PET asdescribed in Experiment 2.

In this experiment, the lead polyglutamated folate DFO-HBPE-NPsdelivered CT20p via PSMA targeting to PCa, facilitating regression ofthe primary tumors and the metastatic lesions in the animal modelsstudied, even in the castration and hormone signaling conditions.Minimal off-target toxicity was observed due to the specific PSMAtargeting to PCa and the fact that CT20p was only cytotoxic to cancercells. On the other hand, off-target toxicity was observed in micetreated with free Doxo, Doxo-s-s-Folate, or Folate HBPE (Doxo).Cytotoxic effects in response to the introduction of CT20p resulted inloss of F-actin and reduced integrin signaling which led to celldetachment and induction of death (e.g. anoikis). These events werefatal for metastatic cancer cells but did not occur in normal cells.

If using polyglutamated folate DFO-HBPE(CT20p) NPs, compared tofolate-HBPE-Doxo, to impair metastasis is poor or the dose is toxic, thedose or delivery scheme can be modulated until metastasis inhibition isobserved or biodistribution PET imaging data can be used to modifytargeting.

For an expected difference in means of at least 75% and a power of 95%,a sample size of 5-8 mice was calculated to account for biologicalvariability in all in vivo experiments. The statistical core wasperformed as described in Experiment 1.

Example 3

In this aspect, a new theranostic (therapeutic and imaging) nanoparticlethat encapsulates a novel cytotoxic peptide, CT20p, for the treatment ofprostate cancer is disclosed. Animal experiments were necessary toexamine the effectiveness of the nanoparticle preparations aftervalidation in in vitro cell culture experiments. Mice used in this studyprovide a model in which to test the cytotoxicity of nanoparticlesloaded with peptides and develop optimal immune responses as follows:

Crl:SHO-Prkdc^(scid)Hr^(hr) mice, males, 8-16 For completion of tasks inweeks of age Experiment 2 = 130 mice CB17.Cg-Prkdc^(scid)Lyst^(bg-J)/Crlmice, males, For completion of tasks in 8-16 weeks of age Experiment 3 =320 mice

Subcutaneous Tumor Implantation and Treatment

5-10 million prostate cancer cells (PC3, LNCaP) were implanted in micesubcutaneously (sc) into the right or left flanks. Mice were monitoreduntil tumors reached 10 to 15 mm² in size. Tumors were measured usingcalipers 2-3 times and ultrasound 2-3 times per week. Upon tumordetection, mice were injected intravenously with nanoparticles andcontrols as described in Experiment 2. In addition, each experiment alsoincluded a PBS control. The amount of nanoparticles delivered to micewere experimentally determined. Tumor size continued to be monitoredafter treatment for up to 21 days if tumors ulcerated. At the end of thestudy, animals were euthanized and tumors and other tissues (kidneys,spleen, liver, lungs and brain) were removed for evaluation. Althoughmice were monitored daily for visual signs of distress, mice were scoredtwo-three times per week, and those showing distress were euthanizedprior to the study endpoint. Mice euthanized prior to end of study werealso dissected and anatomically evaluated.

In Vivo Toxicity and Pharmacokinetic (PK) Studies

Groups of mice (non-tumor) were intravenously treated weekly withescalating doses of nanoparticles (2-20 mg/kg/dose) and the mice wereobserved for changes in weight and food uptake. Blood was collected asdescribed herein and sent for clinical chemistry analysis. After 12-13weeks, mice were euthanized and organs were removed for histologicalanalysis. For PK studies, groups of mice were intravenously treated withnanoparticles over a 24 hours period. Blood was withdrawn after 5, 15,30, 1, 2, 4, 6, 8, 12, 18, and 24 hours and analyzed for clearance ofpeptide (CT20p) and nanoparticles.

Recovery of Serum and Urine

Blood (<0.5% animal body weight/wk) was drawn from and collected into 1ml syringes (containing 3.2% sodium citrate) from the tail vein of mice.Mice were restrained for blood collection and a heat lamp was used whenneeded. Anesthesia is not normally required for this procedure.Following the bleeding procedure, mice were euthanized by CO₂. Othermethods for drawing blood such as (retro-orbital bleed or heartpuncture) can be adapted following the same handling procedure asdescribed herein. Anesthesia (i.e. intraperitoneal administration oftribromoethanol (250 mg/kg, 0.2 ml volume) can be used in the event thatthese alternative methods are employed. For recovery of spontaneousurine, mice were placed in clean cage with plastic wrap and voided urinerecovered. For terminal recovery of urine after mice were euthanized,direct puncture of the bladder with a needle using a syringe.

Implantation of Tumors Cells in Prostate

Male 6-8 week old male mice were used. Pre-surgery pain medication wasinjected according to animal facility's instructions (e.g. 0.1 mg/kgbody weight subcutaneous Buprenex). At the time of surgery, animals wereanesthetized with isoflurane. The lower abdominal region was disinfectedand, using a sterile scalpel or sharpened sterile surgical scissors, alow midline abdominal incision of approximately 3-4 mm was made. Theprostate lobes were identified and a 20 μl volume (˜2.5×105 cells)solution was injected into the prostate gland. The muscle layer wasclosed with sutures and the skin with staples. Mice were imaged weeklyfor up to 4-6 weeks to monitor tumor growth. Treatment groups weretested as described above in Experiment 3.

Bone Tumor Model

Following the procedures described herein, male animals wereanesthetized and cell suspensions were prepared. Injection volume permouse was 20 μl containing 0.2˜1.5×10⁶ PC-3 cells. To determine optimalendpoints, variable numbers of cancer cells were injected intratibiallyand tumor growth was detected by radiographs at appropriate times postinoculation. For intra-tibial implantation of tumor cells, anesthetizedmice were disinjected. The ankle (tibia and fibula) was rotatedlaterally and the knee bent so that the anterior crest of the tibialbody was clearly visible through the skin. The syringe needle wasaligned with the long axis of the tibia and the needle was insertedpercutaneously through the knee joint to place the needle tip on theproximal tuberosity of tibia. Drilling occurred by rotating the syringe(half to ¾ turn). Once the needle tip had advanced to the correctposition, the syringe was released and the syringe stayed still. X-rayimages were used to confirm correct position of the needle within thebony trabeculae near the growth plate. The drilling needle was retractedand the syringe was loaded with the cell suspension. The injectionsyringe needle was placed in the drilled position and suspension wasslowly injected. Tumor growth was followed up by X-ray images.

Monitoring of Tumor Growth

For imaging tumors in mice, mice were anesthetized and fluorescenceimaging was detected with the Carestream Multi Spectral FX imagingstation. Examination of luminescence was performed with the IVIS system.Ultrasound imaging to locate the tumor and perform needle guidedinjections was done using the Visual Sonics Vivo 2100. Followingimaging, mice were observed for recovery and were returned to thehousing room.

Number of Animals

The use of mice in the studies is justified by the extensive database ofinformation available to support studies. SCID mice are routinely usedfor implantation of prostate tumor cells and the murine model of bonemetastatic disease is a well-established system to study tumorinvasions. Numbers of animals used in experiments were determined usinga statistical power analysis based on results achieved in the pilotstudy. A significance level (alpha) of 0.05 (two-tailed) was used. Apower (beta) of 95% was chosen to determine sample size. Statisticalanalysis (GraphPad StatMate, Prism) determined that a sample size of 8mice in each group had a 95% power to detect a difference between theexperimentally determined standard deviation and test values with asignificance level of 5%. All experiments were repeated three times forreproducibility.

Experiment 2 required 48 SCID mice to test the in vivo toxicity of 2-3lead compounds and controls and 88 mice (˜11 time points) to completethe PK studies.

Experiment 3 required 320 mice to develop the castration model and theintra-prostate and intra-tibia orthotropic models as described fortesting of 2-3 lead compounds and controls.

By adding a few mice for biological variability (such as tumors nottaken), a total of ˜480 mice were estimated for the period of 4 years.

Veterinary Care of the Animals

Veterinary care at the University of Central Florida (UCF) transgenicanimal facility at Lake Nona was provided by in-house animal caretechnicians and a licensed veterinarian. The new facility was fullyaccredited by the Association of Assessment and Accreditation of AnimalCare, International (AAALAC) in 2011 and has an approved assurance onfile with the Office of Laboratory Animal Welfare, NIH (OLAW). All micewere housed under pathogen-free conditions. Animal care was provided inaccordance with the procedures outlined in the “Guide for the Care andUse of Laboratory Animals” (NIH Publication No. 86-23, 1985). Animalswere identified by cage card/ear notch. Immunodeficient mice were housedin sterile cages and handled under aseptic conditions.

Mice experienced minimal pain or distress. Mice were placed in acomfortable restraining device for tail vein injections withnanoparticle suspensions. For imaging and the orthotopic model, micewere anesthetized with 2% isoflurane with 1% oxygen in an inductionchanger, and, during the procedure, the anesthetized state wasmaintained with a nose cone. All procedures were performed using thevolatile fluorocarbon, isoflurane, involved a precision vaporizer (whichwas calibrated and certified within 12 months of the experiment asrequired by IACUC policy) in an induction chamber followed by use of anose cone. Depth of anesthesia was confirmed by observing respirationrate and verifying absence of response to ear, toe, and/or tail pinch.Response evaluated included withdrawal as well as an increase or changein respiratory rate and/or pattern.

After the implantation of tumors, mice were observed daily for signs ofdistress and body condition scoring (BCS, see below) used to assessproblems. If tumors were >10% of the mouse's body size (˜1 cm indiameter), became ulcerated or interfered with normal functions, themouse was euthanized.

Example of BCS guidelines are as follows:

5: The mouse is obese, and bones cannot be felt at all;

4: The mouse is well-fleshed, and bones are barely felt;

3: The mouse is in optimal condition. Bones are palpable but notprominent;

2: The mouse is becoming thin and bones are prominent. This category maybe further divided subjectively as +2, 2, and −2. Euthanasia isrecommended for BCS of −2.

1: Muscle wasting is advanced, fat deposits are gone, and bones are veryprominent. Euthanasia is mandatory.

A body condition score of 2 or 1 indicates a decline in overallcondition, and euthanasia is recommended. A weight loss of 10-15% withina few days or an overall weight-loss of 20% is also an indication foreuthanasia.

Animals were euthanized if evidence of pain or distress was evident orif the tumor was greater than 10% of the animal body weight. Animalswere sacrificed before tumor ulceration occurs. Euthanasia wasconsidered for animals exhibiting any of these signs of distress.Euthanasia was performed by CO₂ asphyxiation in an inhalation chamber.This method is consistent with the recommendations of the Panel onEuthanasia of the American Veterinary Medical Association. Mice wereconfirmed to have no signs of responsiveness, respiration or heart beatprior to collection. Death was insured by a second method such asthoracic puncture or cervical dislocation.

Disclosed herein is a novel theranostic approach for breast cancer,using a polymeric nanoparticle carrying DFO-⁸⁹Zr as contrast agent andthe cytotoxic peptide CT20p. Animal experiments were used to evaluatethe theranostic efficacy of the lead preparation after in vitroexperiments. The correlation of the induced changes in T1 from baselinewith the change in tumor volume after dose finding studies was tested.It was determined that a total of 50 animals were needed. SCID beigemice, male animals (since these are prostate tumors), ca. 3 weeks oldwere used.

A sample size calculation based on the difference in means and anexpected 100% difference in means revealed that 6 animals per group wereneeded for a power of 95% and a p-values of 0.05 in the imagingexperiments. There were 2 main sets of animal experiments; these aredetailed in Experiment 2.

Experiment 2 required: For dose finding studies, 3 different doses ineach 6 animals and one control for a total of 24 animals. For imagingstudies, 2 doses of particles and one control was required with 6 miceeach for a total of 18 mice. The total number of animals was therefore42. To adjust for biological variability (such as tumors not taken), atotal of 50 mice were used.

Memorial Sloan-Kettering Cancer Center's animal care and use program isadministered by the Research Animal Resource Center (RARC) as one of thecore facilities of MSKCC. The program has been fully accredited by theAssociation of Assessment and Accreditation of Animal Care,International (AAALAC) since 1967, is registered with the USDA, and hasan approved assurance on file with the Office of Laboratory AnimalWelfare, NIH (OLAW). RARC is staffed by board-certified laboratoryanimal veterinarians and pathologists, veterinary and animal caretechnicians, management, and administrative support staff. Veterinarystaff is available 24 hours a day, 7 days a week to address emergencies.The program is supported by the Laboratory of Comparative Pathology,which provides anatomic and clinical pathologic evaluation of animals,tissues, and fluids in support of animal health and the use of animalmodels.

The animal resource program is housed in three state-of-the-artfacilities occupying a total of 62,500 net ft2 of usable space. Allvivaria contain barrier rodent housing facilities. One also supports thehousing and use of large animal species. Specialized facilities for theuse of animal models exposed to biological and hazardous chemical agentsand for conducting surgical procedures in large and small animals areavailable. Multi-modality imaging suites containing computerizedtomography (CT) scanners, optical instruments (bioluminescence,fluorescence and optical tomography), as well as PET and a SPECT/CTscanner are available for imaging large and/or small animals. Thesescanners are all housed directly within the animal facility in theZuckerman research center. Small animal ultrasound scanner and a 4.7Tand a 7T MRI scanners are also available to image and conductspectroscopic studies in small animal models. Specialized housing roomsfor maintaining aquatic species are also available.

All procedures were performed under inhalational anesthesia using thevolatile fluorocarbon isoflurane, administered using a precisionvaporizer (which was calibrated and certified within 12 months of theexperiment as required by IACUC policy) in an induction chamber followedby use of a nose cone. Waste anesthetic gas was scavenged by using anactivated carbon canister or by working under a fume hood, scavengingsnorkel, or a biological safety cabinet equipped with an activatedcarbon filter. Depth of anesthesia was confirmed by observingrespiration rate and verifying absence of response to ear, toe, and/ortail pinch. Response evaluated included withdrawal as well as anincrease or change in respiratory rate and/or pattern.

While on treatment or after having tumor implanted, mice were monitoredat least every other day for evidence of toxicity: pain, morbidity, lossof body weight (>10%), dehydration, poor grooming and/or excessive tumorburden resulting from tumor implantation or treatment. If required, painwas alleviated with buprenorphine injected subcutaneously as needed,under guidance of the veterinarian service.

Animals were euthanized if evidence of pain or distress was evident orif the tumor was greater than 10% of the animal body weight. Animalswere sacrificed before tumor ulceration occurred. Euthanasia wasconsidered for animals exhibiting any of these signs of distress.Euthanasia was performed by CO₂ asphyxiation in an inhalation chamber.This method is consistent with the recommendations of the Panel onEuthanasia of the American Veterinary Medical Association.

What is claimed is:
 1. A method of identifying a solid tumor cell target, comprising, 1) contacting a cell with an effective amount of a composition comprising at least one nanoparticle conjugated with a targeting ligand that is a substrate for a solid tumor-specific cell protein, wherein the nanoparticle further comprises an imaging compound; 2) identifying one or more nanoparticles bound to the cells by using imaging devices; and optionally, 3) monitoring the solid tumor cell target by repeating 1) and 2).
 2. The method of claim 1, further comprising treating the solid tumor cell by killing or inhibiting its growth.
 3. The method of claim 1 or 2, wherein the solid tumor cell is a prostate cancer cell.
 4. The method of claim 3, wherein the prostate cancer cell is castration resistant prostate cancer.
 5. The method of claim 1 or 2, wherein the solid tumor cell is a breast cancer cell.
 6. The method of claim 1 or 2, wherein the solid tumor cell is a colon cancer cell.
 7. The method of claim 1 or 2, wherein the solid tumor cell is a pancreas cancer cell.
 8. The method of claim 1 or 2, wherein the solid tumor cell is a lung cancer cell.
 9. The method of claim 1 or 2, wherein the nanoparticle further comprises, in its hydrophobic interior, a therapeutic agent.
 10. The method of claim 9, wherein the therapeutic agent is CT20p or a mutant CT20 peptide.
 11. The method of claim 9, wherein the therapeutic agent is a mitotoxic peptide.
 12. The method of claim 9, wherein the therapeutic agent is an anti-metastatic agent.
 13. The method of claim 9, wherein the therapeutic agent is an antiandrogenic agent.
 14. The method of claim 8, wherein the therapeutic agent is an anti-neoplastic agent.
 15. The method of claim 1, 2 or 9, wherein the solid tumor-specific cell protein is prostate specific membrane antigen (PSMA).
 16. The method of claim 1, 2 or 9, wherein the nanoparticles are polymeric nanoparticles.
 17. The method of claim 1, 2 or 9, wherein the nanoparticles are hyperbranched polyester nanoparticles (HBPE-NPs).
 18. The method of claim 1, 2 or 9, wherein the targeting ligand is a folate compound.
 19. The method of claim 1, 2 or 9, wherein the targeting ligand is a glutamate compound.
 20. The method of claim 1, 2 or 9, wherein the targeting ligand is a polyglutamated folate compound.
 21. The method of claim 1 or 2 or 9, wherein the targeting ligand is glutamate azido urea.
 22. The method of claim 1, 2 or 9, wherein the targeting ligand is folate azido urea.
 23. The method of claim 1, 2 or 9, wherein the targeting ligand is glutamate azido urea.
 24. The method of claim 1, 2 or 9, wherein the targeting ligand is a bifunctional glutamate-folate hybridized compound.
 25. The method of claim 1, 2 or 9, wherein the nanoparticle comprises a chelating ligand such as desferrioxamine (DFO).
 26. The method of claim 1, 2 or 9, wherein the imaging compound is a PET detectable compound.
 27. The method of claim 1, 2 or 9, wherein the PET detectable compound is ⁸⁹Zr.
 28. The method of claim 1, 2 or 9, wherein the PET detectable compound is CU or other PET detectable compounds.
 29. The method of claim 1, 2 or 9, wherein the composition comprises a polyglutamated folate-HBPE-DFO[CT20p]-nanoparticles.
 30. The method of claim 1, 2 or 9, wherein the nanoparticle further comprises PEG.
 31. The method of claim 1, 2 or 9, wherein the targeting ligand is at high density/valency.
 32. The method of claim 1, 2 or 9, wherein the targeting ligand is at low density/valency.
 33. A cancer therapeutic composition, comprising: at least one nanoparticle conjugated with a targeting ligand that is a substrate for a solid tumor-specific cell protein, wherein the nanoparticle further comprises an imaging compound and having a therapeutic agent encapsulated in the hydrophobic interior of the nanoparticle.
 34. The composition of claim 33, wherein the therapeutic agent is CT20p or a mutant CT20 peptide.
 35. The composition of claim 33, wherein the therapeutic agent is a mitotoxic peptide.
 36. The composition of claim 33, wherein the therapeutic agent is an anti-metastatic agent.
 37. The composition of claim 33, wherein the therapeutic agent is an antiandrogenic agent.
 38. The composition of claim 33, wherein the therapeutic agent is an anti-neoplastic agent.
 39. The composition of claim 33, wherein the solid tumor-specific cell protein is prostate specific membrane antigen (PSMA).
 40. The composition of claim 33, wherein the nanoparticles are polymeric nanoparticles.
 41. The composition of claim 33, wherein the nanoparticles are hyperbranched polyester nanoparticles (HBPE-NPs).
 42. The composition of claim 33, wherein the targeting ligand is a folate compound.
 43. The composition of claim 33 wherein the targeting ligand is a glutamate compound.
 44. The composition of claim 33, wherein the targeting ligand is a polyglutamated folate compound.
 45. The composition of claim 33, wherein the targeting ligand is glutamate azido urea.
 46. The composition of claim 33, wherein the targeting ligand is folate azido urea.
 47. The composition of claim 33, wherein the targeting ligand is glutamate azido urea
 48. The composition of claim 33, wherein the targeting ligand is a bifunctional glutamate-folate hybridized compound.
 49. The composition of claim 33, wherein the nanoparticle comprises a chelating ligand such as desferrioxamine (DFO).
 50. The composition of claim 33, wherein the imaging compound is a PET detectable compound.
 51. The composition of claim 33, wherein the PET detectable compound is ⁸⁹Zr.
 52. The composition of claim 33, wherein the PET detectable compound is Cu or other PET detectable compounds.
 53. The composition of claim 33, wherein the composition comprises a polyglutamated folate-HBPE-DFO[CT20p]-nanoparticles.
 54. The composition of claim 33, wherein the nanoparticle further comprises PEG.
 55. The composition of claim 33, wherein the targeting ligand is at high density/valency.
 56. The composition of claim 33, wherein the targeting ligand is at low density/valency.
 57. A method for treating cancer, comprising: a) administering to a subject diagnosed with prostate cancer an effective amount of a nanoparticle composition comprising, at least one nanoparticle conjugated with targeting ligand that is a substrate for a solid tumor-specific cell protein, wherein the nanoparticle further comprises an imaging compound and has a therapeutic agent encapsulated in the hydrophobic interior of the nanoparticle.
 58. The method of claim 57, wherein the cancer is prostate cancer.
 59. The method of claim 58, wherein the prostate cancer is castration resistant prostate cancer.
 60. The method of claim 57, wherein the cancer is breast cancer.
 61. The method of claim 57, wherein the cancer is colon cancer.
 62. The method of claim 57, wherein the cancer is pancreas cancer.
 63. The method of claim 57, wherein the cancer is lung cancer.
 64. The method of claim 57, wherein the therapeutic agent is CT20p or a mutant CT20 peptide.
 65. The method of claim 57, wherein the therapeutic agent is a mitotoxic peptide.
 66. The method of claim 57, wherein the therapeutic agent is an anti-metastatic agent.
 67. The method of claim 57, wherein the therapeutic agent is an antiandrogenic agent.
 68. The method of claim 57, wherein the therapeutic agent is an anti-neoplastic agent.
 69. The method of claim 57 wherein the solid tumor-specific cell protein is prostate specific membrane antigen (PSMA).
 70. The method of claim 57, wherein the nanoparticles are polymeric nanoparticles.
 71. The method of claim 57, wherein the nanoparticles are hyperbranched polyester nanoparticles (HBPE-NPs).
 72. The method of claim 57, wherein the targeting ligand is a folate compound.
 73. The method of claim 57, wherein the targeting ligand is a glutamate compound.
 74. The method of claim 57, wherein the targeting ligand is a polyglutamated folate compound.
 75. The method of claim 57, wherein the targeting ligand is glutamate azido urea.
 76. The method of claim 57, wherein the targeting ligand is folate azido urea.
 77. The method of claim 57, wherein the targeting ligand is glutamate azido urea
 78. The method of claim 57, wherein the targeting ligand is a bifunctional glutamate-folate hybridized compound.
 79. The method of claim 57, wherein the nanoparticle comprises a chelating ligand such as desferrioxamine (DFO).
 80. The method of claim 57, wherein the imaging compound is a PET detectable compound.
 81. The method of claim 57, wherein the PET detectable compound is ⁸⁹Zr.
 82. The method of claim 57, wherein the PET detectable compound is CU or other PET detectable compounds.
 83. The method of claim 57, wherein the composition comprises a polyglutamated folate-HBPE-DFO[CT20p]-nanoparticles.
 84. The method of claim 57, wherein the nanoparticle further comprises PEG.
 85. The method of claim 57, wherein the targeting ligand is at high density/valency.
 86. The method of claim 57, wherein the targeting ligand is at low density/valency.
 87. The method of claim 57, further comprising administering another therapeutic or radiolabeled compound.
 88. A nanoparticle, comprising: a polymeric nanoparticle conjugated with targeting ligand that is a substrate for a solid tumor-specific cell protein, wherein the nanoparticle further comprises one or more imaging compounds and/or one or more therapeutic agents encapsulated in the hydrophobic interior of the nanoparticle.
 89. The nanoparticle of claim 88, wherein the therapeutic agent is CT20p or a mutant CT20 peptide.
 90. The nanoparticle of claim 88, wherein the therapeutic agent is a mitotoxic peptide.
 91. The nanoparticle of claim 88, wherein the therapeutic agent is an anti-metastatic agent.
 92. The nanoparticle of claim 88, wherein the therapeutic agent is an antiandrogenic agent.
 93. The nanoparticle of claim 88, wherein the therapeutic agent is an anti-neoplastic agent.
 94. The nanoparticle of claim 88, wherein the solid tumor-specific cell protein is prostate specific membrane antigen (PSMA).
 95. The nanoparticle of claim 88, wherein the nanoparticles are polymeric nanoparticles.
 96. The nanoparticle of claim 88, wherein the nanoparticles are hyperbranched polyester nanoparticles (HBPE-NPs).
 97. The nanoparticle of claim 88, wherein the targeting ligand is a folate compound.
 98. The nanoparticle of claim 88, wherein the targeting ligand is a glutamate compound.
 99. The nanoparticle of claim 88, wherein the targeting ligand is a polyglutamated folate compound.
 100. The nanoparticle of claim 88, wherein the targeting ligand is glutamate azido urea.
 101. The nanoparticle of claim 88, wherein the targeting ligand is folate azido urea.
 102. The nanoparticle of claim 88, wherein the targeting ligand is glutamate azido urea
 103. The nanoparticle of claim 88, wherein the targeting ligand is a bifunctional glutamate-folate hybridized compound.
 104. The nanoparticle of claim 88, wherein the nanoparticle comprises a chelating ligand such as desferrioxamine (DFO).
 105. The nanoparticle of claim 88, wherein the imaging compound is a PET detectable compound.
 106. The nanoparticle of claim 88, wherein the PET detectable compound is ⁸⁹Zr.
 107. The nanoparticle of claim 88, wherein the PET detectable compound is CU or other PET detectable compounds.
 108. The nanoparticle of claim 88, wherein the nanoparticle comprises a polyglutamated folate-HBPE-DFO[CT20p]-nanoparticles.
 109. The nanoparticle of claim 88, wherein the nanoparticle further comprises PEG.
 110. The nanoparticle of claim 88, wherein the targeting ligand is at high density/valency.
 111. The nanoparticle of claim 88, wherein the targeting ligand is at low density/valency.
 112. A nanoparticle comprising a hyperbranched hyperbranched polyester functionalized with azide groups.
 113. A cancer therapeutic composition, comprising: at least one nanoparticle conjugated with a targeting ligand that is a substrate for a solid tumor-specific cell protein, wherein the nanoparticle further comprises one or more therapeutic agents encapsulated in the hydrophobic interior of the nanoparticle.
 114. The composition of claim 113, wherein the nanoparticle comprises more than one therapeutic agents.
 115. The composition of claim 113, wherein the therapeutic agent is CT20p or a mutant CT20 peptide.
 116. The composition of claim 113, wherein the therapeutic agent is a mitotoxic peptide.
 117. The composition of claim 113, wherein the therapeutic agent is an anti-metastatic agent.
 118. The composition of claim 113, wherein the therapeutic agent is an antiandrogenic agent.
 119. The composition of claim 113, wherein the therapeutic agent is an anti-neoplastic agent.
 120. The composition of claim 113, wherein the solid tumor-specific cell protein is prostate specific membrane antigen (PSMA).
 121. The composition of claim 113, wherein the nanoparticles are polymeric nanoparticles.
 122. The composition of claim 113, wherein the nanoparticles are hyperbranched polyester nanoparticles (HBPE-NPs).
 123. The composition of claim 113, wherein the targeting ligand is a folate compound.
 124. The composition of claim 113 wherein the targeting ligand is a glutamate compound.
 125. The composition of claim 113, wherein the targeting ligand is a polyglutamated folate compound.
 126. The composition of claim 113, wherein the targeting ligand is glutamate azido urea.
 127. The composition of claim 113, wherein the targeting ligand is folate azido urea.
 128. The composition of claim 113, wherein the targeting ligand is glutamate azido urea
 129. The composition of claim 113, wherein the targeting ligand is a bifunctional glutamate-folate hybridized compound.
 130. The composition of claim 113, wherein the nanoparticle comprises a chelating ligand such as desferrioxamine (DFO).
 131. The composition of claim 113, wherein the nanoparticle further comprises PEG.
 132. The composition of claim 113, wherein the targeting ligand is at high density/valency.
 133. The composition of claim 113, wherein the targeting ligand is at low density/valency.
 134. The composition of claim 114, wherein the therapeutic agents are independently chosen from a DNA intercalator, topoisomerase inhibitor, microtubule stabilizer (taxol), receptor kinase inhibitor, kinase inhibitor, aromatase inhibitor, and anti-androgen. 